Use of difluoromethylornithine (DFMO) for the treatment of amyotrophic lateral sclerosis

ABSTRACT

The present invention provides methods and compositions for modulating polyamine pathway activity as a means for ameliorating neurodegenarative disorders. In particular, for ameliorating the symptoms or onset of amyotrophic lateral sclerosis (ALS) by modulating the gene and protein products involved the polyamine pathway, such as by inhibiting the enzyme, ornithine decarboxylase (ODC), involved in the synthesis of the polyamine, putrescine. Compositions and methods are disclosed for inhibiting the polyamine pathway producing lower polyamine levels resulting in a beneficial effect on ALS. This can be accomplished by using modulating agents such as analogs, or polyamine analogs, and antiproliferative drugs. In particular, the ODC inhibitor difuoromethylornithine (DFMO) is disclosed as a useful pharmacological agent in the modulation and treatment of ALS. Screening assays for pharmacological agents that are capable of decreasing polyamine levels and/or reducing cell proliferation are also disclosed.

RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S.Ser. No. 60/333,263, filed Nov. 16, 2001 entitled “Methods forMonitoring and Treating Amyotrophic Lateral Sclerosis”.

BACKGROUND OF THE INVENTION

[0002] The technical field of the invention concerns methods andcompositions for the treatment of neurodegenerative diseases, such asAmyotrophic Lateral Sclerosis (ALS).

[0003] Neurodegencrative diseases are generally characterized by adegeneration of neurons in either the brain or the nervous system of anindividual. In addition to ALS, various other diseases, such asHuntington's disease, Parkinson's disease, Alzheimer's disease andMultiple Sclerosis, fall within this category. These diseases aredebilitating and the damage that they cause is often irreversible.Moreover, in the case of a number of these diseases, the outcome isinvariably fatal.

[0004] Progress is being made on many fronts to find agents that canarrest the progress of these diseases. Nonetheless, the presenttherapies for most, if not all, of these diseases provide very littlerelief.

[0005] Accordingly, a need exists to develop therapies that can alterthe course of neurodegenerative diseases or, in the case of diseaseslike ALS, prolong the survival time of patients with such diseases. Moregenerally, a need exists for better methods and compositions for thetreatment of neurodegenerative diseases in order to improve the qualityof the lives of those afflicted by such diseases.

SUMMARY OF INVENTION

[0006] The present invention provides methods and compositions formodulating polyamine pathway activity as a means for amelioratingneurodegenarative disorders, in particular, for ameliorating thesymptoms or onset of amyotrophic lateral sclerosis (ALS). ALS has beenassociated with increased polyamine levels. In particular, the inventionprovides an approach to circumvent the present limitations of ALStherapy by modulating the gene and protein products involved thepolyamine pathway. For example, by modifying the level of polyaminessuch as putrescine, by inhibiting the enzyme ornithine decarboxylase(ODC). Other polyamines that are involved in the polyamine pathway thatcan be modulated include, but are not limited to spermidine, andspermine. The method and composition of the invention alter polyamineconcentrations or levels such that the alteration provides a protectiveeffect in a subject with ALS. This protective effect manifests in theincrease in longevity of the subject, as well as to slow or arrest theprogress of the disease.

[0007] The proteasome is the biological machinery that is responsiblefor normal degradation of proteins is also involved in the polyaminepathway. The proteasome damage associated with neurological disordersmay lead to an increased half life of ornithine decarboxylase (ODC), theenzyme that converts ornithine into the polymamine putrescine. Thisincrease in ODC yields an increased amount of putrescine andsubsequently increased levels of spermine and spermidine. Compositionsand methods are disclosed for inhibiting the polyamine pathway producinglower polyamine levels resulting in a beneficial effect on ALS. This canbe accomplished by using modulating agents based analogs, or polyamineanalogs, that can be used to inhibit the pathway. Substrate mimics ofputrescine, spermidine, and spermine would act by blocking the polyaminepathway resulting in downregulation of the pathway, producing decreasedlevels of polyamines. At least one of the enzymes of the polyaminepathway should be inhibited. The enzyme inhibition may be reversible ornonreversible. In a preferred embodiment, ornithine decarboxylase (ODC)is inhibited leading to a reduction in putrescine levels. Alternatively,proliferation of microglia and astrocytes, which has been associatedwith neurological disorders can be decreased by administering atherapeutically effective amount of an antiproliferative drug, such ashydroxyurea, difuoromethylornithine (DFMO), and various polyamineanalogs.

[0008] Accordingly, in one aspect, the invention provides a method formodulating polyamine pathway activity in a subject comprisingadministrating a therapeutically effective amount of adifuoromethylornithine (DFMO) to the subject. The DFMO can be a racemicmixture of (D)- and (L)-DFMO, (D)-DFMO, or (L)-DFMO.

[0009] In another aspect, the invention provides a method of inhibitingprogression of amyotrophic lateral sclerosis (ALS) by administering to asubject thereof a pharmaceutical composition comprising atherapeutically effective amount of difuoromethylornithine (DFMO), orpharmaceutically acceptable salts thereof, to thereby modulate thepolyamine pathway and ameliorate the progression of ALS. The DFMO can beadministered via any appropriate route, such as an oral route and anintravenous route. The amount of DFMO can be determined based on thesize and disease condition of the subject, for example, the DMFO can beadministered in an amount of between 100 mg/Kg/day and 10,000 mg/Kg/day.Preferably, DFMO can be administered in an amount between 1000 mg/Kg/dayand 7000 mg/Kg/day. More preferably, DFMO can be administered in anamount between 2000 mg/Kg/day and 5000 mg/Kg/day. Most preferably, DFMOcan be administered in an amount between 3000 mg/Kg/day and 4000mg/Kg/day. The amount of DFMO can be adjusted based on patient responseor plasma levels of DFMO.

[0010] The methods and compositions of the invention may also be used todetect if a subject has ALS based on the difference in polyamine levelsin test sample from a subject compared with a control sample.Accordingly, in another aspect, the invention pertains to methods fordetecting amyotrophic lateral sclerosis (ALS), or monitoring theprogression of ALS in a subject by measuring a polyamine level in afirst sample from a subject, measuring the polyamine level in a secondsample; and comparing the difference in the level of the polyamine inthe first and second samples, wherein a difference in the level of thepolyamine is an indicator for ALS.

[0011] To test whether the subject has ALS, the first sample can be atest sample from a subject, and the second sample can be control samplewith normal levels of the polyamine. The difference or deviation in thepolyamine levels between the test sample and the control sampleindicates that a person has ALS. For example, increased levels of thepolyamine, putrescine. although a difference in the level of anypolyamine involved in the polyamine pathway may be used in the assay,such as a difference in the level of spermidine and spermine.

[0012] The assay may also be used to screen for the progression of ALSby monitoring the difference in polyamine levels at different timepoints in a subject with ALS. This may be accomplished for example, byactivating astrocytes to proliferation, adding a pharmacological agent,and monitoring the rate of astrocyte proliferation. Pharmacologicalagents that inhibit microglia and/or astrocyte proliferation may beuseful candidates for further testing in vivo.

[0013] Accordingly, in one aspect, the invention pertains to a method toscreen for the progression of ALS by monitoring the difference inpolyamine levels at different time points in a subject with ALS. Thiscan be performed by taking sample from a subject, or testingpharmacological agents in vitro with neurological cell lines. When thefirst sample is a sample from a subject taken at a first time point, andthe second sample is sample from a subject taken at a second time point.The second time point can be a day, a week, a month, and so forth, untila compendium of data is available with the different levels of thepolyamine to monitor the progression of ALS. Also within the scope ofthe invention, is an assay that measures more than one polyamine, forexample, the levels of putrescine and/or spermidine or spermine.

[0014] The level of polyamine can be assessed by measuring theexpression, or the activity of at least one of the enzymes involved inthe polyamine pathways, these include, but are not limited to, ornithinedecarboxylase, S-adenosylmethionine decarboxylase, and arginase.Alternatively, the level of polyamine is assessed by measuring theexpression or activity of polyamine itself.

[0015] In yet another aspect, the invention pertains to a method forscreening for a pharmacological agent that modulates the polyaminepathway in a subject with amyotrophic lateral sclerosis (ALS). Thisassay can be performed by measuring a level of a polyamine in a samplefrom a subject at a first point, administering a test pharmacologicalagent to a subject, monitoring the level of the polyamine in a samplefrom a subject at discrete times points following administration, andcomparing the difference in the level of the polyamine to determine ifthe pharmacological agent changes the level of the polyamine. Thepolyamine to be measured includes, but is not limited to, putrescine,spermidine, and spermine. Alternatively, more than one polyamine may bedetected.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a scheme depicting the polyamine pathway;

[0017]FIG. 2 is a graph showing the effect of discontinuous DFMOdelivery on survival of female SOD1 G93A mice;

[0018]FIG. 3 is a graph showing the effect of discontinuous DFMOdelivery on the neurological progression of female SOD1 G93A mice;

[0019]FIGS. 4A and B are graphs showing repeat studies on the effect ofcontinuous DFMO delivery on survival of a combined group of female andmale SOD1 G93A mice;

[0020]FIGS. 5A and B are graphs of repeat studies showing the effect ofcontinuous DFMO delivery on the neurological progression of a combinedgroup of female and male SOD1 G93A mice;

[0021]FIGS. 6A and B are graphs of repeat studies showing the effect ofcontinuous DFMO delivery on survival of male SOD1 G93A mice;

[0022]FIGS. 7A and B are graphs of repeat studies showing the effect ofcontinuous DFMO delivery on the neurological progression of male SOD1G93A mice;

[0023]FIGS. 8A and B are graphs of repeat studies showing the effect ofcontinuous DFMO delivery on survival of female SOD1 G93A mice;

[0024]FIGS. 9A and B are graphs of repeat studies showing the effect ofcontinuous DFMO delivery on the neurological progression of female SOD1G93A mice;

[0025]FIG. 10 is a graph showing the effect of hydroxyurea, ananti-proliferative agent with ribonucleotide reductase inhibitoractivity, on survival of female SOD1 G93A mice;

[0026]FIG. 11 is a graph showing the effect of hydroxyurea, ananti-proliferative agent with ribonucleotide reductase inhibitoractivity, on ALS disease progression of female SOD1 G93A mice;

[0027]FIG. 12 is a graph showing the effect of DENSPM, a polyamineanalog with anti-proliferative activity, on survival of female SOD1 G93Amice; and

[0028]FIG. 13 is a graph showing the effect of DENSPM, a polyamineanalog with anti-proliferative activity, on ALS disease progression offemale SOD1 G93A mice.

DETAILED DESCRIPTION

[0029] So that the invention is more clearly understood, the followingterms are defined:

[0030] The terms “neurological disorder” and “neurodegenerativedisorder,” as used interchangeably herein refer to an impairment orabsence of a normal neurological function or presence of an abnormalneurological function in a subject. For example, neurological disorderscan be the result of disease, injury, and/or aging. As used herein,neurological disorder also includes neurodegeneration which causesmorphological and/or functional abnormality of a neural cell or apopulation of neural cells. Non-limiting examples of morphological andfunctional abnormalities include physical deterioration and/or death ofneural cells, abnormal growth patterns of neural cells, abnormalities inthe physical connection between neural cells, under- or over productionof a substance or substances, e.g., a neurotransmitter, by neural cells,failure of neural cells to produce a substance or substances which itnormally produces, production of substances, e.g., neurotransmitters,and/or transmission of electrical impulses in abnormal patterns or atabnormal times. Neurodegeneration can occur in any area of the brain ofa subject and is seen with many disorders including, for example,Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, Huntington'sdisease, Parkinson's disease, and Alzheimer's disease.

[0031] “Amyotrophic lateral sclerosis” or “ALS” are terms understood inthe art and as used herein to denote a progressive neurodegenerativedisease that affects upper motor neurons (motor neurons in the brain)and/or lower motor neurons (motor neurons in the spinal cord) andresults in motor neuron death. As used herein, the term “ALS” includesall of the classifications of ALS known in the art, including, but notlimited to classical ALS (typically affecting both lower and upper motorneurons), Primary Lateral Sclerosis (PLS, typically affecting only theupper motor neurons), Progressive Bulbar Palsy (PBP or Bulbar Onset, aversion of ALS that typically begins with difficulties swallowing,chewing and speaking), Progressive Muscular Atrophy (PMA, typicallyaffecting only the lower motor neurons) and familial ALS (a geneticversion of ALS).

[0032] The term “subject,” as used herein, refers to any living organismcapable of eliciting an immune response. The term subject includes, butis not limited to, humans, nonhuman primates such as chimpanzees andother apes and monkey species; farm animals such as cattle, sheep, pigs,goats and horses; domestic mammals such as dogs and cats; laboratoryanimals including rodents such as mice, rats and guinea pigs, and thelike. The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be covered.

[0033] The term “pharmacological agent,” as used herein, refers to thecompound, or compounds, that are used to modulate polyamine levels,astrocyte, microglia and/or macrophage proliferation in a subjectafflicted with a neurodegenerative disease. Exemplary pharmacologicalagents according to the present invention are the compounds DFMO,hydroxyurea, and DENSPM. Other pharmacological agents include analogsand variants of the compound DFMO, as well as all intermediate compoundsand intermediate processes in the creation the compound DFMO. The term“pharmacological agent” is also intended to include other ODC inhibitorsas well as inhibitor and analogs of the polyamine pathway. Non-limitingexamples of irreversible ODC inhibitors include DFMO, α-difluoromethylornithine, α-halomethyl ornithine, methyl and ethyl esters ofmonofluoromethyl dehydroornithine, the R, R-isomer of methyl acetylenicputrescine (i.e., (2R, 5R)-6-heptyne-2,5-diamine), optical isomers andcombinations thereof. In addition, pharmacological agents is alsointended to include other ODC inhibitors with similar formula andfunction to DFMO that are described by the core formulas:

[0034] where X is —CHF₂ or —CH₂F, R is H or COR₁, R₁ is OH or loweralkoxy groups, and the pharmaceutical acceptable salts and isomersthereof. Other inhibitors of ODC known in the art are described in U.S.Pat. No. 4,499,072, U.S. Pat. No. 5,002,879; and the work of Bey et al.(“Inhibition of Basic Amino Acid Decarboxylases Involved in PolyamineBiosynthesis,” Inhibition of Metabolism Biological Significance andBasis for New Therapies, McCann et al, eds.; Academic Press, (1987)1-32).

[0035] The terms “DFMO,” and “Eflornithine” are used interchangeably andrefer to the compound that is chemically designated as2-(Difuoromethyl)-DL-ornithine, 2-(Difluoromethyl)ornithine,DL-α-difuoromethylornithine, N-Difuoromethylornithine, ornidyl, andα,δ-Diamino-α-(difluoromethyl)valeric acid, has a molecular formula ofC₆H₁₂F₂N₂O₂, has a molecular weight of 182.17, and has the followingchemical structure:

[0036] The term “DFMO” is intended to cover all isotopes of the abovecompound. DFMO is intended to cover all pharmaceutically acceptablesalts and/or isomeric forms. Optically pure preparations ofα-difluoromethylornithine, having either the (D)-configuration aroundthe alpha carbon of the molecule or the (L)-configuration around thealpha carbon, as well as racemic mixtures are encompassed by the term“DFMO.” Racemic and optically pure forms of DFMO can be preparedaccording to known methods described in U.S. Pat. No. 4,413,141, U.S.Pat. No. 4,399,151, U.S. Pat. No. 4,438,270, U.S. Pat. No. 4,560,795,U.S. Pat. No. 4,743,691, U.S. Pat. No. 4,866,206, EP 357029 AZ which arehereby incorporated by reference. The steady state plasma concentrationof DFMO can be determined using methods known in the art as described inU.S. Pat. No. 6,277,411, Smithers (Pharm. Res. 5, 684-686 (1988));Bitonti et al. (Biochem. Pharmacol. 35, 351-354 (1986)); and Grove etal. (J. Chromatogr. 223, 409-416 (1981)) which are hereby incorporatedby reference.

[0037] The term “polyamine”, is a well-understood term of art, andrefers to any group of aliphatic, straight-chain amines derivedbiosynthetically from amino acids; polyamines are reviewed in Marton etal. (Ann. Rev. Pharm. Toxicol. 35:55-91 (1995)). By “polyamine” isgenerally meant a naturally-occurring polyamine or natural polyamine,which are naturally produced in cukaryotic cells. Examples of polyaminesinclude putrescine, spermidine, spermine and cadaverine.

[0038] The term “polyamine analog,” as used herein, refers to an organicaction structurally similar but non-identical to naturally-occurringpolyamines such as spermine and/or spermidine and their diamineprecursor, putrescine. Polyamine analogs can be branched or unbranched,or incorporate cyclic moieties. Examples of polyamine analogs include,without limitation, N¹, N¹⁴-diethylhomo-spermine (DEHSPM) and N¹,N¹²-diethylspermine (DESPM). See, for example, WO 98/17624 and U.S. Pat.5,541,320. U.S. Patent Nos. 5,037,846 and 5,242,947 disclose polyaminescomprising primary amino groups. In some embodiments, polyamine analogsinclude those wherein all nitrogen atoms of said polyamine analogs areindependently secondary, tertiary, or quartenary amino groups.

[0039] The term “DENSPM,” as used herein, refers to the compound, orcompounds, that is chemically designated as 1,3-Propanediamine,N,N′-bis(3-(ethylamino)propyl)- or N(1),N(11)-diethylnorspermine, has amolecular formula of C₁₃H₃₂N₄, and has the following chemical structure:

[0040] The term “DENSPM” is intended to cover all isotopes andmetabolites of the above compound. DENSPM is intended to cover allpharmaceutically acceptable salts and/or isomeric forms as well asstructurally similar compounds. Non-limiting examples of derivatives canbe found in U.S. Pat. No. 5,962,533, U.S. Pat. No. 5,886,051, U.S. Pat.No. 6,184,232, U.S. Pat. No. 6,342,534, and U.S. Pat. No. 6,235,794.

[0041] The term “alkyl” as used herein refers to a cyclic, branched, orstraight chain chemical group containing carbon and hydrogen, such asmethyl, butyl, t-butyl, pentyl, cyclopropyl, and octyl. Alkyl groups canbe either unsubstituted or substituted with one or more subsituents,e.g., halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy,benzyl. Alkyl groups can be saturated or unsaturated (e.g., containing—C═C— or —C C— subunits), at one or several positions. Unless otherwisespecified, alkyl groups will comprise 1 to 8 carbon atoms, preferably 1to 6, and more preferably 1 to 4 carbon atoms. “Cycloalkyl” refers tocyclic alkyl groups only, such as cyclopropyl, cyclobutyl, cyclopentyl,etc. “n-alkyl” refers to a linear (i.e., straight-chain) alkyl grouponly, while “branched alkyl” refers to branched alkyl groups to theexclusion of cyclic and linear alkyl groups. “Alkenyl” refers to acyclic, branched, or straight chain chemical group containing carbon andhydrogen where at least one bond is monounsaturated, such as ethenyl,cyclopentenyl, or 1,3-butadienyl. Alkenyl groups can be substituted asindicated for alkyl groups. Alkenyl groups can be designated as cyclic,linear (n-alkenyl) or branched in an analogous fashion to the precedingdesignations for alkyl. An “aryl” is an unsaturated aromatic carbocyclicgroup having a single ring (e.g., phenyl), or multiple condensed rings(e.g., naphthyl), which can optionally be unsubstituted or substitutedwith amino, hydroxyl, alkyl, alkoxy, chloro, halo, mercapto and othersubstituents.

[0042] The term “stercoisomer” as used herein refers to an opticalisomer of a compound, including enantiomers and diastereomers. Unlessotherwise indicated, structural formula of compounds are intended toembrace all possible sterioisomers.

[0043] The term “salt” as used herein refers to a compound formed by thereplacement of one or more hydrogen atoms with elements or groups, whichis composed of anions and cations, which usually ionizes in water; asalt is formed, for instance, by neutralization of an acid by a base. Apolyamine analog salt can comprise, for example, chloride ions.

[0044] The phrase “protected derivative” as used herein refers to acompound protected with a protecting group. “Protecting group” refers toa chemical group that exhibits the following characteristics: 1) reactsselectively with the desired functionality in good yield (preferably atleast 80%, more preferably at least 90%, more preferably at least 95%,still more preferably at least 99%) to give a protected substrate thatis stable to the projected reactions for which protection is desired; 2)is selectively removable from the protected substrate to yield thedesired functionality; and 3) is removable in good yield (preferably atleast 80%, more preferably at least 90%, more preferably at least 95%,still more preferably at least 99%) by reagents compatible with theother functional group(s) present or generated in such projectedreactions. Examples of suitable protecting groups can be found in Greeneet al. (1991) Protective Groups in Organic Synthesis, 2^(nd) Ed. (JohnWiley & Sons, Inc., New York). Exemplary protecting groups for the aminofunctionality include, but are not limited to, mesitylenesulfonyl(MesSO2), benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc),t-butyldimethylsilyl (TBDIMS), 9-fluroenylmethyloxycaronyl (Fmoc), orsuitable photolabile protecting groups such as 6-nitroveratryloxycarbonyl (Nvoc).

[0045] The term “modulating agent” as used herein refers to a compoundthat alters at least one step in the polyamine pathway such that thealteration in the polyamine pathway produces a modification in the aneurodegenerative disorder. In particular, the alteration in thepolyamine pathway produces an amelioration of ALS and the symptoms ofALS, such as increasing the life expectancy of the subject. The term“modulating agent” includes polyamine analogs, inhibitors that target atleast one enzyme in the polyamine pathway, and activators of anantizyme. In one embodiment, the modulating agent targets a specificenzyme involved in the pathway, for example, a modulating agent thatinterferes with one of the enzymes involved in that pathway such as ODC.The modulation to the pathway can be in the form of increasing,decreasing, elevation, or depressing processes or signal transductioncascades, involving a target gene or a target protein, e.g., ODC. Thismodulation may result by direct, (e.g., direct binding) or indirect(e.g., use of analogs that mimic the action of the native substrate orbind to the enzyme substrate complex) interaction with the targetprotein. The modifications can result in a direct affect on the targetprotein, e.g., inhibition of ODC. Alternatively, the modifications canbe indirect modification of a process or cascade involving the targetprotein, e.g., inhibition of ODC which reduces the concentration ofputrescine, which subsequently alters the concentration of spermidineand spermine.

[0046] Examples of modulating agents that target specific enzymesinclude, but are not limited to, ODC inhibitors and polyamine analogs.Examples of ODC inhibitors include, but are not limited to, ornithinedecarboxylase (ODC) inhibitor is selected from the group consisting ofdifuoromethylornithine (DFMO), α-halomethyl ornithine, methyl ester ofmonofluoromethyl dehydroornithine, ethyl ester of monofluoromethyldehydroornithine, and R, R-isomer of methyl acetylenic putrescine.Examples of polyamine analogs include, but are not limited to, selectedfrom the group consisting of 1,11-bis(ethyl)norspermine(1,11-bis(ethylamino)-4,8-diazaundecane), 1,8-bis(ethyl)spermindine,1,12-bis(ethyl)spermine, 1,14-bis(ethylamino)-5,10-diazatetradecane and1,19-bis(ethylamino)-5,10,15-triazanonadecane. In another embodiment,the modulating agent targets more that one step in the polyaminesynthesis, such as an agent that effects both ODC and spermidinesynthase or spermine synthase. In another yet another embodiment, morethan one modulating agent can be used to alter the polyamine pathway.For example, a combination of modulating agents comprising at least onemodulating agent that alters ODC activity and at least one modulatingagent that alters spermidine synthase activity or spermine synthaseactivity. In yet another embodiment, the combination of modulatingagents can comprise at least one modulating agent that is a polyamineanalog, and at least one modulating agent that is an inhibitor of anenzyme sinvolved in the polyamine pathway.

[0047] Examples of the amelioration of the symptoms of ALS in a subjectinclude, but are not limited to, prolonging the life expectancy of asubject, prevention of onset of the disease, slowing or arrestingdisease progression (as measured by neurological examination or othermeans, such as MRI, FVC, MUNE etc.), altering the state of microglia andastrocytes in a subject with ALS, improving muscle weakness in asubject, (e.g., improving weakness in the hands, arms, legs), improvingswallowing or breathing, improving twitching (fasciculation) andcramping of muscles, improving the use of the limbs in a subject.

[0048] The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of thepharmacological agent may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thepharmacological agent to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the pharmacological agent are outweighed by thetherapeutically beneficial effects.

[0049] The term “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0050] The invention is described in more detail in the followingsubsections:

[0051] I. Neurodegenerative Diseases

[0052] Regional alterations in polyamines and polyamine metabolism arenoted in many neurodegenerative diseases such as Creutzfelt Jacob's(CJD), Huntington's (HD), Stroke and Alzheimer's disease (AD). Inaddition, over expression of ornithine decarboxylase (ODC) had beenobserved under basal conditions in the Wobbler mouse, one of the animalmodels of ALS. Human ALS patients show elevated levels of ornithine (thepolyamine precursor) in the spinal tissue and arginase (the enzyme whichconverts arginine to ornithine) in the cerebrospinal fluid (CSF).Polyamine dysregulation may be the cause of many neurodegenativeconditions, some of which are described below:

[0053] (a) Huntington's Disease

[0054] Huntington's disease is a hereditary disorder caused by thedegeneration of neurons in certain areas of the brain. This degenerationis genetically programmed to occur in certain areas of the brain,including the cells of the basal ganglia, the structures that areresponsible for coordinating movement. Within the basal ganglia,Huntington's disease specifically targets nerve cells in the striatum,as well as cells of the cortex, or outer surface of the brain, whichcontrol thought, perception and memory. Neuron degeneration due toHuntington's disease can result in uncontrolled movements, loss ofintellectual capacity and faculties, and emotional disturbance, such as,for example, mood swings or uncharacteristic irritability or depression.

[0055] As discussed above, neuron degeneration due to Huntington'sdisease is genetically programmed to occur in certain areas of thebrain. Studies have shown that Huntington's disease is caused by agenetic defect on chromosome 4, and in particular, people withHuntington's disease have an abnormal repetition of the genetic sequenceCAG in the Huntington's disease gene, which has been termed IT15. TheIT15 gene is located on the short arm of chromosome 4 and encodes aprotein called huntingtin. Exon I of the IT15 gene contains apolymorphic stretch of consecutive glutamine residues, known as thepolyglutamine tract (D. Rubinsztein, “Lessons from Animal Models ofHuntington's Disease,” TRENDS in Genetics, 18(4): 202-9 (April 2002)).Asymptomatic individuals typically contain fewer than 35 CAG repeats inthe polyglutamine tract.

[0056] (b) Multiple Sclerosis

[0057] Multiple Sclerosis (MS) is a chronic disease that ischaracterized by “attacks,” during which areas of white matter of thecentral nervous system, known as plaques, become inflamed. Inflammationof these areas of plaque is followed by destruction of myelin, the fattysubstance that forms a sheath or covering that insulates nerve cellfibers in the brain and spinal cord. Myelin facilitates the smooth,high-speed transmission of electrochemical messages between the brain,spinal cord, and the rest of the body. Damage to the myelin sheath canslow or completely block the transmission of these electrochemicalmessages, which can result in diminished or lost bodily function.

[0058] The most common course of Multiple Sclerosis manifests itself asa series of attacks, which are followed by either complete or partialremission, during which the symptoms lessen only to return at some laterpoint in time. This type of MS is commonly referred to as“relapsing-remitting MS.” Another form of MS, called“primary-progressive MS,” is characterized by a gradual decline into thedisease state, with no distinct remissions and only temporary plateausor minor relief from the symptoms. A third form of MS, known as“secondary-progressive MS,” starts as a relapsing-remitting course, butlater deteriorates into a primary-progressive course of MS.

[0059] The symptoms of MS can be mild or severe, acute or of a longduration, and may appear in various combinations. These symptoms caninclude vision problems such as blurred or double vision, red-greencolor distortion, or even blindness in one eye, muscle weakness in theextremities, coordination and balance problems, muscle spasticity,muscle fatigue, paresthesias, fleeting abnormal sensory feelings such asnumbness, prickling, or “pins and needles” sensations, and in the worstcases, partial or complete paralysis. About half of the people sufferingfrom MS also experience cognitive impairments, such as for example, poorconcentration, attention, memory and/or judgment. These cognitivesymptoms occur when lesions develop in those areas of the brain that areresponsible for information processing.

[0060] (c) Alzheimer's Disease

[0061] Alzheimer's disease is a progressive, neurodegenerative diseasethat affects the portions of the brain that control thought, memory andlanguage. This disease is characterized by progressive dementia thateventually results in substantial impairment of both cognition andbehavior. The disease manifests itself by the presence of abnormalextracellular protein deposits in brain tissue, known as “amyloidplaques,” and tangled bundles of fibers accumulated within the neurons,known as “neurofibrillary tangles,” and by the loss of neuronal cells.The areas of the brain affected by Alzheimer's disease can vary, but theareas most commonly affected include the association cortical and limbicregions. Symptoms of Alzheimer's disease include memory loss,deterioration of language skills, impaired visuospatial skills, andimpaired judgment, yet those suffering from Alzheimer's retain motorfunction.

[0062] (d) Parkinson's Disease

[0063] Parkinson's disease is a motor system disorder caused by the lossof nerve cells, or neurons, found in the substantia nigra region of themid-brain. These neurons produce dopamine, a chemical messenger moleculethat is found in the brain and helps control or direct muscle activity.Dopamine is used by the cells of the substantia nigra as aneurotransmitter to signal other nerve cells. Parkinson's disease occurswhen these neurons die or become impaired, thereby decreasing dopaminelevels within the brain. Loss of dopamine causes the neurons to fireuncontrollably, which leaves patients unable to direct or control theirbodily movement in a normal manner. The four main symptoms ofParkinson's disease are trembling in the hands, arms, legs, jaw andface; stiffness of the limbs and/or trunk; a slowness of movement,referred to as bradykinesia; and impaired balance and/or coordination.Parkinson's disease is both chronic, i.e., it persists over a longperiod of time, and progressive, i.e., the symptoms grow worse overtime.

[0064] (e) Amyotrophic Lateral Sclerosis

[0065] Amyotrophic Lateral Sclerosis (ALS) is a universally fatalneurodegenerative condition in which patients progressively lose allmotor function - unable to walk, speak, or breathe on their own, ALSpatients die within two to five years of diagnosis. The incidence of ALSincreases substantially in the fifth decade of life. Evidence isaccumulating that as a result of the normal aging process the bodyincreasingly loses the ability to adequately degrade mutated ormisfolded proteins. The proteasome is the piece of biological machineryresponsible for most normal degradation of proteins inside cells. Agerelated loss of function or change of function of the proteasome is nowthought to be at the heart of many neurodegenerative conditions,including Alzheimer's disease, Parkinson's disease, Huntington'sdisease, and ALS.

[0066] Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig'sdisease, is a fatal neurodegenerative disease affecting motor neurons ofthe cortex, brain stem and spinal cord. (Hirano, A., “Neuropathology ofALS: an overview,” Neurology, 47(4 Suppl. 2): S63-6 (1996)). Onset ofALS occurs in the fourth or fifth decade of life (median age of onset is57) and is fatal within two to five years after diagnosis (Williams, D.B. and A. J. Windebank, “Motor neuron disease (amyotrophic lateralsclerosis),” Mayo Clin. Proc., 66(1): 54-82 (1991)). ALS affectsapproximately 30,000 Americans with nearly 8,000 deaths reported in theUS each year. ALS remains one of the most devastating diseases andadvances in treatment are desperately needed.

[0067] The cardinal feature of ALS is the loss of spinal motor neurons,which causes the muscles under their control to weaken and waste awayleading to paralysis. ALS has both familial (5-10%) and sporadic formsand the familial forms have now been linked to several distinct geneticloci (Deng, H. X., et al., “Two novel SOD1 mutations in patients withfamilial amyotrophic lateral sclerosis,” Hum. Mol. Genet., 4(6): 1113-16(1995); Siddique, T. and A. Hentati, “Familial amyotrophic lateralsclerosis,” Clin. Neurosci., 3(6): 338-47(1995); Siddique, T., et al.,“Familial amyotrophic lateral sclerosis,” J Neural Transm. Suppl., 49:219-33(1997); Ben Hamida, et al., “Hereditary motor system diseases(chronic juvenile amyotrophic lateral sclerosis). Conditions combining abilateral pyramidal syndrome with limb and bulbar amyotrophy,” Brain,113(2): 347-63 (1990); Yang, Y., et al., “The gene encoding alsin, aprotein with three guanine-nucleotide exchange factor domains, ismutated in a form of recessive amyotrophic lateral sclerosis,” Nat.Genet., 29(2): 160-65 (2001); Hadano, S., et al., “A gene encoding aputative GTPase regulator is mutated in familial amyotrophic lateralsclerosis 2,” Nat. Genet., 29(2): 166-73 (2001)). About 15-20% offamilial cases are due to mutations in the gene encoding Cu/Znsuperoxide dismutase 1 (SOD1) (Siddique, T., et al., “Linkage of a genecausing familial amyotrophic lateral sclerosis to chromosome 21 andevidence of genetic-locus heterogeneity,” N. Engl. J. Med., 324(20):1381-84 (1991); Rosen, D. R., et al., “Mutations in Cu/Zn superoxidedismutase gene are associated with familial amyotrophic lateralsclerosis.” Nature, 362(6415): 59-62 (1993)).

[0068] Although a great deal is known about the pathology of ALS littleis known about the pathogenesis of the sporadic form and about thecausative properties of mutant SOD protein in familial ALS (Bruijn, L.I. and D. W. Cleveland, “Mechanisms of selective motor neuron death inALS: insights from transgenic mouse models of motor neuron disease,”Neuropathol. Appl. Neurobiol., 22(5): 373-87 (1996); Bruijn, L. I., etal., “Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutantindependent from wild-type SOD1,” Science, 281(5384): 1851-54 (1998)).Many models have been speculated, including glutamate toxicity, hypoxia,oxidative stress, protein aggregates, neurofilament and mitochondrialdysfunction, however, no model has comprehensively described the diseasepathogenesis (Cleveland, D. W., et al., “Toxic mutants in Charcot'ssclerosis,” Nature, 378(6555): 342-43 (1995); Cleveland, D. W., et al.,“Mechanisms of selective motor neuron death in transgenic mouse modelsof motor neuron disease,” Neurology, 47(4 Suppl. 2): S54-61, discussionS61-2(1996); Cleveland, D. W., “From Charcot to SOD1: mechanisms ofselective motor neuron death in ALS,” Neuron, 24(3): 515-20 (1999);Cleveland, D. W. and J. D. Rothstein, “From Charcot to Lou Gehrig:deciphering selective motor neuron death in ALS,” Nat. Rev. Neurosci.,2(11): 806-19 (2001); Couillard-Despres, S., et al., “Protective effectof neurofilament heavy gene overexpression in motor neuron diseaseinduced by mutant superoxide dismutase,” Proc. Natl. Acad. Sci. USA,95(16): 9626-30 (1998); Mitsumoto, H., “Riluzole—what is its impact inour treatment and understanding of amyotrophic lateral sclerosis?” Ann.Pharmacother., 31(6): 779-81 (1997); Skene, J. P. and D. W. Cleveland,“Hypoxia and Lou Gehrig,” Nat. Genet., 28(2): 107-8 (2001); Williamson,T. L., et al., “Toxicity of ALS-linked SOD1 mutants,” Science,288(5465): 399 (2000)).

[0069] Amyotrophic lateral sclerosis (ALS), a progressive, degenerativedisease of the voluntary motor system, attacks motor neurons in thecortex, brain stem and spinal cord, and the progressive degeneration ofthese nerve cells often leads to their death (A. Hirano, “Neuropathologyof ALS: an overview,” Neurology 47:4(S2): S63-66 (1996); See e.g., L PRowland, Merritt's Textbook of Neurology, ed. L P Rowland, Hereditaryand acquired motor neuron disease (Philadelphia: Williams and Wilkins,1995)). As motor neurons die, they lose the ability to stimulate musclefibers, and consequently, the brain loses the ability to initiate andcontrol muscle movement. In later stages of the disease, patients becometotally paralyzed, yet retain their cognitive functioning.

[0070] Early symptoms of ALS include increasing muscle weakness,particularly in the arms and legs, and in the muscles associated withspeech, swallowing and breathing. Symptoms of weakness and muscleatrophy usually begin asymmetrically and distally in one limb, and thenspread within the neuroaxis to involve contiguous groups of motorneurons. Symptoms can begin either in bulbar or limb muscles. Clinicalsigns of both lower and upper motor neuron involvement are required fora definitive diagnosis of ALS. Respiration is usually affected late inlimb onset patients, but occasionally can be an early manifestation inpatients with bulbar onset symptoms. ALS is a universally fatalneurodegenerative condition.

[0071] Although the etiology of the disease is unknown, the dominanttheory is that neuronal cell death in ALS is the result ofover-excitement of neuronal cells due to excess extracellular glutamate.Glutamate is a neurotransmitter that is released by glutaminergicneurons, and is taken up into glial cells where it is converted intoglutamine by the enzyme glutamine synthetase, glutamine then re-entersthe neurons and is hydrolyzed by glutaminase to form glutamate, thusreplenishing the neurotransmitter pool. In a normal spinal cord andbrain stem, the level of extracellular glutamate is kept at lowmicromolar levels in the extracellular fluid because glial cells, whichfunction in part to support neurons, use the excitatory amino acidtransporter type 2 (EAAT2) protein to absorb glutamate immediately. Adeficiency in the normal EAAT2 protein in patients with ALS, wasidentified as being important in the pathology of the disease (See e.g.,Meyer et al., J. Neurol. Neurosurg. Psychiatry, 65: 594-596 (1998); Aokiet al., Ann. Neurol. 43: 645-653 (1998); Bristol et al., Ann Neurol. 39:676-679 (1996)). One explanation for the reduced levels of EAAT2 is thatEAAT2 is spliced aberrantly (Lin et al., Neuron, 20: 589-602 (1998)).The aberrant splicing produces a splice variant with a deletion of 45 to107 amino acids located in the C-terminal region of the EAAT2 protein(Meyer et al., Neureosci Lett. 241: 68-70 (1998)). Due to the lack of,or defectiveness of EAAT2, extracellular glutamate accumulates, causingneurons to fire continuously. The accumulation of glutamate has a toxiceffect on neuronal cells because continual firing of the neurons leadsto early cell death.

[0072] Studies by Gurney showed that the TgN (SOD1-G93A) G1H mice, anestablished animal model for ALS drug screening, showed significantlyincreased numbers of activated astrocytes (P<0.01) at 100 days of age inboth the cervical and lumbar spinal cord regions (Hall ED, O. J., GurneyM E., “Relationship of microglial and astrocytic activation to diseaseonset and progression in a transgenic model of familial ALS,” Glia,23(3): 249-256 (1998)). However, at 120 days of age, the activation loststatistical significance. In contrast, microglial activation wassignificantly increased several-fold at both 100 and 120 days. Geneexpression analysis of post mortem ALS spinal cord demonstratedsignificant increase in macrophage/microglial activation markers ascompared to normal and other disease controls (Malaspina A, et al.,“Differential expression of 14 genes in amyotrophic lateral sclerosisspinal cord detected using gridded cDNA arrays,” J. Neurochem., 77(1):132-45 (2001); Gullans, “Gene expression profiling in human ALS spinalcord,” 2000). In addition, recent studies based on gene expressionanalysis of mSOD1 mouse spinal cord at various stages of ALSdemonstrated the presence of activated microglial signature well beforeclinical changes (70 days) suggesting that microglial activation occurprior to neuronal damage (Rothstein, J., “Gene expression profiling inmSOD G93A mice,” 2000; Olsen, M. K., et al., “Disease mechanismsrevealed by transcription profiling in SOD1-G93A transgenic mouse spinalcord,” Ann. Neurol., 50(6): 730-40 (2001)). Therapies used for relapsingmultiple sclerosis (MS), including interferon (IFN) beta-1b, IFNbeta-1a, and glatiramer acetate (Dhib-Jalbut S. “Mechanisms of action ofinterferons and glatiramer acetate in multiple sclerosis.” Neurology(2002) 58(8 Suppl 4):S3-9) may also be useful in combination therapy forALS. In MS therapy, IFNs bind to cell surface-specific receptors,initiating signaling pathways, which end with the secretion ofantiviral, antiproliferative, and immunomodulatory gene products. Theseantiproliferative gene products may have beneficial effects in thetreatment of ALS.

[0073] Presently, there is no cure for ALS, nor is there a therapy thathas been proven effective to prevent or reverse the course of thedisease. Several drugs have recently been approved by the Food and DrugAdministration (FDA). To date, attempts to treat ALS have involvedtreating neuronal degeneration with long-chain fatty alcohols which havecytoprotective effects (See U.S. Pat. No. 5,135,956); or with a salt ofpyruvic acid (See U.S. Pat. No. 5,395,822); and using a glutaminesynthetase to block the glutamate cascade (See U.S. Pat. No. 5,906,976).For example, Riluzole™, a glutamate release inhibitor, has been approvedin the U.S. for the treatment of ALS, and appears to extend the life ofat least some patients with ALS. However, some reports have indicatedthat even though Riluzole™ therapy can prolong survival time, it doesnot appear to provide an improvement of muscular strength in thepatients. Therefore, the effect of Riluzole™ is limited in that thetherapy does not modify the quality of life for the patient(Borras-Blasco et al., Rev. Neurol., 27: 1021-1027 (1998)).

[0074] (f) Prion-Associated Diseases

[0075] The prion protein (PrP) is closely associated with a group offatal neurodegenerative diseases (Ma, J. and Lindquist, S., “Wild-typePrP and a mutant associated with prion disease are subject to retrogradetransport and proteasome degredation,” Proc. Natl. Acad Sci.,98(26):14955-14960 (2001)). This group of disorders is characterized byvacuolation of the brain's gray matter, also known as spongioformchange. These diseases can take a variety of forms. For example, thesediseases can be sporadic, dominantly heritable, as well as transmissibledisorders. In humans, the most prevalent form of prion disease isCreutzfeldt-Jakob disease, while in animals, the most comnon form isknown as scrapie. Other disorders in this group include kuru,Gerstmann-Straussler-Scheinker disease and fetal familial insomnia. Alldisorders are invariably fatal.

[0076] In particular, the symptoms of Creutzfeldt-Jakob disease includea rapidly progressive deterioration of intellectual abilities (alsoknown as dementia). The median duration of this illness, from on-set ofsymptoms to death is around four months. As the disease stateprogresses, the dementia is typically accompanied by other symptoms suchas ataxia, muscular rigidity, and spontaneous and irregular limb jerks,also known as myoclonus.

[0077] (g) Spinocerebellar Ataxia

[0078] Ataxias are diseases wherein a person loses the ability tocoordinate muscle activity during voluntary muscle contraction, andtherefore, loses the ability to coordinate smooth bodily movements.Spinocerebellar ataxia is the most common form of hereditary ataxia.Symptoms of the on-set of spinocerebellar ataxia include limb ataxia,nystagmus (rhythmical oscillation of the eyeballs, in either a pendularor jerky motion), kyphoscoliosis (a deformity of the spine characterizedby extensive flexion), and pes cavus (a contracted foot, or exaggerationof the normal arch of the foot). The major pathological changes thatoccur with the disease state occur in the posterior columns of thespinal cord. Spinocerebellar ataxia is most often an autosomal recessiveinherited disorder.

[0079] (h) Spinomuscular Atrophy

[0080] Spinomuscular atrophy (SMA) is a disease of the anterior horncells of the spinal cord. There are several different types of SMA,including Type I or Acute (Severe) SMA, which is also known asWerdnig-Hoffmann Disease, Type II (Chronic) SMA, Type III (Mild) SMA,often referred to as Kugelberg-Welander or Juvenile SMA, Type IV (AdultOnset) SMA, and Adult Onset X-Linked SMA, also known as Kennedy'sSyndrome or Bulbo-Spinal Muscular Atrophy, which occurs in males, butfemales may be carriers. SMA affects the voluntary muscles that areresponsible for activities such as crawling, walking, head and neckcontrol, and swallowing. SMA mainly affects the proximal muscles, or themuscles closes to the trunk of a person's body. Symptoms includeweakness in the legs and arms, with weakness in the legs being greaterthan weakness in the arms. Other symptoms may include tonguefasciculations, or abnormal movements of the tongue. During the courseof SMA, however, a person's senses, feelings and intellectual activityremain unaffected.

[0081] II. Polyamine Pathway Modulating Pharmacological Agents

[0082] Regionally altered polyamine metabolism is noted in manyneurodegenerative diseases such as Creutzfelt Jacob's (CJD),Huntington's (HD), Stroke and Alzheimer's disease (AD). The Wobblermouse, which is used as a motor neuron disease model, has been shown tohave, increased spinal expression of ornithine decarboxylase (ODC), therate-limiting enzyme in polyamine biosynthesis. Ornithine, the substrateof ODC, has been shown to be substantially elevated (300%) in the spinaltissue of human ALS patients. Arginase, the enzyme responsible for thesynthesis of ornithine, is over-expressed two to eight fold in the CSFof human patients. The present invention provides methods and assaysthat relate polyamine dysregulation to neurological disorders such asALS. In addition, screening assays for pharmacological agents thatcapable of reducing polyamine dyregulation are provided by the presentinvention.

[0083] (a) The Polyamine Pathway

[0084] The polyamines, putrescine, spermidine, and spermine, are a groupof multivalent organic cationic cell components present in all livingcells. The polyamines play important roles in many fundamental cellularprocesses including the regulation of cell proliferation, celldifferentiation, signaling immune cell activation, transformation, andapoptosis. The majority of signal transduction pathways intersectpolyamine biosynthesis pathways and/or those pathways which regulateintracellular polyamine levels. While their exact roles in theseprocesses are still being explored, these processes are putativelyrelated to the unique charge distribution and flexibility of thesecationic polyamines. They affect these processes by regulatingintracellular signals, chromatin structure, replication, transcriptionand translation. More recently they are thought to even regulate ionchannels. Polyamines can directly bind to DNA and modulate DNA-proteininteractions. These functions may be directly related to polyamines'role in cell proliferation as well as cell death. The production ofhydrogen peroxide during polyamine catabolism may directly influencecell death (Thomas et al. Cell. Mol. Life Sci. 58: 244-258 (2001)). Dueto the fundamental role polyamines play in cell function, cells havedeveloped redundant mechanisms for the regulation of polyamines rangingfrom multiple synthetic pathways to extra cellular uptake mechanisms.The synthesis, uptake, and steady state of the three major polyamines,putrescine, spermidine, and spermine, are tightly regulated attranscriptional, translational and breakdown levels.

[0085] Ornithine, derived from the amino acid arginine as part of theurea cycle, is one of the starting materials for polyamine biosynthesis.Putrescine is synthesized from ornithine through the action of theenzyme ornithine decarboxylase (ODC). The higher polyamines, spennidineand spermine, respectively, are produced via the sequential addition ofaminopropyl groups from decarboxylated adomet (See FIG. 1) catalyzed byS-adenosyl methionine decarboxylase (SAMDC), spermidine and sperminesynthase. Polyamines are found in millimolar concentrations in mammaliancells. The level of polyamines vary between gender (Pegg et al. Am. J.Physiol. 243: C212-C221 (1982)).

[0086] (b) Modulation of the Polyamine Pathway

[0087] Polyamine dysregulation is observed in a variety of diseases.Ornithine decarboxylase, the rate-limiting enzyme in polyaminemetabolism is regulated transcriptionally and post-translationally.Post-translational stability of ODC is regulated by the 26S proteasomethrough regulated degradation of ODC by antizyme. ODC is also a uniqueprotein, which is not degraded through the ubiquitination pathway thatoccurs in the degradation of most proteins. Proteasomal dysfunction isimplicated in many of the neurodegenerative diseases described above andin ALS. Studies in yeast have shown that proteasomal mutants show anincreased half-life of ODC as compared to wild-type cells. This suggeststhat proteasomal dysfunction can increase ODC half-life and increase thepolyamine pool in defective cells. Amplification or up regulation of ODCis observed during carcinogenic events and in hyperproliferativediseases. ODC is under expressed in inflammatory Crohn's disease.Polyamine inhibition is beneficial in some autoimmune systemic lupuserythematosus (SLE) disease and inflammation models. Inhibitors ofpolyamine biosynthesis, polyamine analogs, and oligonucleotide/polyamineanalog combinations have been shown have a positive effect in thereduction, treatment, and prevention of cancer.

[0088] Polyamines are regulated at several levels including synthesis,degradation, uptake and efflux. Modification of polyamine levels at anyone of these levels, such that neurological disorders are affected, iswithin the scope of the present invention. The rapid turnover rate ofODC is positively regulated by polyamines. Additionally cells have atransport mechanism to uptake exogenous polyamines. ODC, S-adenosylmethionine decarboxylase (SAMDC), and spermidine/spermine acetyltransferase (SSAT) may be regulated externally at the transcriptionlevel and may be regulated by polyamines at the translation level.Altering an interacting signal transduction pathway may be capable ofdecreasing the level of polyamines ultimately decreasing neurologicaldisorders and hence are within the scope of the present invention.

[0089] Polyamine levels are also tightly regulated by a complex ofproduct feedback systems. ODC is sensitive to slight changes inspermidine and spermine levels, but is relatively insensitive to itsimmediate product putrescine (Hayashi et al. Biochem 306: 1-10 (1995);(Mitchell et al. Biochim. Biophys. Acta 840: 309-316 (1985)). As littleas 50 μM spermidine in the culture medium can inhibit ODC production forhours. The present invention associates high levels of putrescine withneurological disorders. Thus, one embodiment involves the use ofspermidine or spermidine analogs which are capable of negativelyregulating ODC as discussed below.

[0090] The increase in ODC activity may be related to decreasedproteasomal function. Proteasomal activity decreases with age.Conversely ODC activity increases with age. The average age of ALS onsetis 55 years of age. Considering the declining proteasomal functionconcurrent with increased ODC activity in aging, a causativerelationship may exist between proteasome alteration and ODCstabilization. This relationship may be exploited leading to newtherapies for neurological disorders.

[0091] Ornithine decarboxylase (ODC), the rate limiting step in thebiosynthesis of polyamines from ornithine, has been shown to benegatively regulated by an antizyme, through a non-ubiquination mediateddegradation by the 26S proteasome in an ATP-dependent manner (Murakamiet al. Biochem. J. 304: 183-187 (1993)). The 26S proteasome irreversiblyinactivates ODC, possibly through unfolding, prior to its degradation.The level of antizyme (AZ) is regulated translationally by polyamineload through an unusual translational frameshifting process. To date,three antizymes have been discovered that have an effect on polyaminelevels (Ivanov et al. Proc. Natl. Acad. Sci. USA 97: 4808-4813 (2000)).Degradation of ODC consists of a multistep sequence, includingrecognition, sequestration, unfolding, translocation, and proteasomemediated degradation. Functional studies (Coffino, P. Biochimie 83:319-23 (2001)) have identified regions of ODC and AZ that are requiredfor ODC degradation. A region on the surface of an alpha-beta barrelforming one domain of the ODC monomer has been shown to be required forAZ binding. The carboxyl-terminal region of ODC, exposed by interactionwith AZ, is thought to play a role in ODC recognition by the 26Sproteasome ((Murakami Y et al. Biochem Biophys Res Commun 267:1-6(2000)) and (Andreassen et al. Exp. Neurol. 168: 419-424 (2001))). Whilethe carboxy-terminal half of AZ is sufficient for binding to ODC, anadditional domain located within the AZ amino terminus must be presentfor stimulation of ODC degradation by the proteasome (Coffino, P.Biochimie 83: 319-23 (2001)).

[0092] Modification of any of the multiple steps in the sequence leadingto ODC degradation may lead to decreased levels of ODC (Mitchell J L etal. Biochem Soc Trans 26:591-595 (1998)). By upregulating or increasingthe lifetime of at least one of these antizymes, leading to a decreasein ODC and lower levels of polyamines, neurological disorders may betreated and/or reduced. Oligoamine constructs as well asconformationally constrained analogs of the polyamines have been shownto stimulate antizyme synthesis (Mitchell J L et al. Biochem J. 366:663-671 (2002)). Polyamine analogs that are capable modulating antizymeproduction, including but not limited to, bisethylnorspermine,bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane,may used in the treatment of neurological disorders. Furthermore,studies indicate N-methyl-D-aspartate (NMDA) receptors may also play arole in ODC modulation (Reed L J et al. J Neurochem 55: 780-787 (1990)).

[0093] To study the role of the polyamines and ODC in ALS, polyaminelevels can be analyzed in the early and late stage SOD1 G93A mice. Inaddition, the alterations in other polyamines and ODC in ALS are beinginvestigated. Although increased polyamines are detected in ALS mousebrains, its role in neurodegeneration is not clear. To test thetherapeutic implication of this pathway in ALS, the efficacy ofpolyamine analogs, ornithine analogs, and ODC inhibitors, the SOD1 G93Amice can be screened using the assays provided by the present invention.The ODC inhibitor Eflornithine (DFMO) treated animals show signs ofimproved survival (See Examples 2 and 3). These data demonstrate atarget in ALS that can be exploited for drug development.

[0094] Polyamine deregulation in the SOD1 G93A mouse model may berelated to alterations in proteasomal activity. Studies in yeast haveshown that proteasomal dysfunction can lead to decreased degradation ofODC, changing its half life from a few minutes to a few hours.Proteasome mediated polyamine up regulation could lead not only toproliferation of astrocytes and microglia but could also induce neuronalapoptosis by driving neurons into cell cycle. Additionally polyamines inmicroglia can induce release of inflammation mediators such as reactiveoxygen species (ROS) and pro-inflammatory cytokines.

[0095] The basal activity of ornithine decarboxylase (ODC) was found tobe higher in the spine of Wobbler mice, a model for neurodegenerativedisease, than in control animals (M. Gonzalez Deniselle et al. J SteroidBiochem Mol Biol. 60(3-4):205-13 (1997)). This increased ODC activitymay be associated with astrocytosis within the spinal cord of theWobbler mice since ODC activity may originate in astrocytes (Cintra etal. Neurosci. Lett. 76: 149-153 (1987)). Wobbler mice also showedintense proliferation of astrocytes immunoreactive (ir) for glialfibrillary acidic protein (GFAP) in grey and white matter of the spinalcord. GFAP-ir astrocytes have also been found in the brains of ALSpatients (Murayama et al. Acta Neuropathol. (Berl.) 82: 456-461 (1991))Thus, astrocytosis and increased ODC activity may represent the glialand neuronal response which leads to neurodegeneration. In addition,microgliosis and activation of microglia has been seen inneurodegnerative diseases. The observed microgliosis may be related toincreased ODC expression which leads to killing of neurons by microglia.The present invention provides methods and compositions for modulating,treating, reducing, and/or slowing neurodegeneration, e.g., ALS, byinhibiting the polyamine pathway and/or cell proliferation. In apreferred embodiment, the polyamine pathway is inhibited through theadministration of ODC inhibitors. In another embodiment, microgliosisand/or astrocytosis is inhibited through the administration of ODCinhibitors. In another embodiment, a step in the polyamine pathway canbe inhibited to modulate, treat, reduce, and/or slow neurodegeneration,e.g., ALS, through the administration of at least one of the groupcomprising, polyamine analogs, Adomet analogs, adomet decarboxylaseinhibitors (i.e. methylglyoxylbis (guanylhydrazone)), ODC inhibitors,and arginase inhibitors. In yet another embodiment, more than one enzymein the polyamine pathway can be simultaneously and specificallysuppressed resulting in a synergistic effect. Various natural andchemical inhibitors of the polyamine pathway are reported in theliterature and described below.

[0096] (c) Polyamine Synthesis Inhibitors

[0097] i) Polyamine Analogs

[0098] Compounds that mimic the structure of the polyamines, but inhibitthe pathway can be used in the present invention to lower polyaminelevels in a subject with a neurological disorder. The polyamine analogscan be designed to alter the polyamine methylene backbone changing thecharge distribution of the analog. The charge and length of themethylene bridges between the cations has been showed to be important tothe biological function of the polyamines. Thus, these altered polyamineanalogs can block the polyamine pathway leading to decreased production.Examples of polyamine analogs are described in U.S. Pat. Nos. 6,235,794and 6,172,261. Polyamine analogs have also been shown to be effective asantiproliferative agents (Bergeron et al. J Med Chem 44(15): 2451-2459(2001)).

[0099] The polyamine analogs used in the present invention includecompounds of the formulas 1, 2, 3, 4, and 5, and the correspondingstereoisomers, salts, and protected derivatives thereof:

[0100] where R₁, R₂, R₄, R₆ and R₇ are independently selected from thegroup consisting of hydrogen, alkyl and aryl, and where R₃ and R₅ arealkyl groups;

[0101] where R₁, R₂, R₄, R₆, R₈, and R₉ are independently selected fromthe group consisting of hydrogen, alkyl and aryl, and where R₃, R₅, andR₇ are alkyl groups;

[0102] where R₁, R₂, R₄, R₆, R₈, R₁₀ and R₁₁ are independently selectedfrom the group consisting of hydrogen, alkyl and aryl, and where R₃, R₅,R₇ and R₉ are alkyl groups;

[0103] where R₁ and R₅ are independently selected from the groupconsisting of methyl, ethyl, n-propyl, and isopropyl; where R₂, R₃, andR₄ are independently selected from the group consisting of C₁-C₆ alkyl,C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆ cycloalkyl-C₁-C₆alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀ aryl-C₁-C₆ alkyl; and whereR₆, R₇, R₈ and R₉ are independently selected from the group consistingof H, methyl, and ethyl;

[0104] where R₁ and R₆ are independently selected from the groupconsisting of methyl, ethyl, n-propyl, and isopropyl; where R₂, R₃, R₄and R₅ are independently selected from the group consisting of C₁-C₆alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀ aryl-C₁-C₆alkyl; and where R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected fromthe group consisting of H, methyl, and ethyl.

[0105] In some embodiments, the polyamine analogs will include compoundsof the formula 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and where R₄, R₆ and R₈ arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and further whereR₂ and R₁₁ are hydrogen atoms.

[0106] In some embodiments, the polyamine analogs will include compoundsof the formula 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and where R₄, R₆ and R8 arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and where R₂ andR₁₁ are hydrogen atoms, and further where the polyamine analogs have amolecular weight less than 500.

[0107] In some embodiments, compounds also include compounds of theformula 4, where R₆, R₇, R₈ and R₉ are H; where R₁ and R₅ are ethyl;where R₆, R₇, R₈ and R₉ are H and R₁ and R₅ are ethyl; and/or where R₂and R₄ are independently selected from the group consisting of C₁-C₆alkyl and R₃ is independently selected from the group consisting ofC₁-C₆ alkyl, C₃-C₆ alkenyl, C₁-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀ aryl-C₁-C₆alkyl.

[0108] Additional polyamine analogs useful in the present inventioninclude compounds of the formula 6, and the corresponding stereoisomers,salts, and protected derivatives thereof:

[0109] where R₄ is C₂-C₆ n-alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkenyl, or C₃-C₆ aryl; R₃ and R₅ are independently chosen from asingle bond, C₁-C₆ alkyl, or C₁-C₆ alkenyl; R2 and R₆ are independentlychosen from C₁-C₆ alkyl, C₁-C₆ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkenyl, or C₃-C₆ aryl; R₁ and R₇ are independently chosen from H,C₁-C₆ alkyl, or C₂-C₆ alkenyl; and R₈, R₉, R₁₀, and R₁₁, are H.

[0110] In some embodiments of the compounds of formula 6, R₁ and R₇ areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl.

[0111] Additional polyamine analogs useful in the present inventioninclude compounds of the formula 7, and the corresponding stereoisomers,salts, and protected derivatives thereof:

[0112] where R₄ is C₁-C₆ n-alkyl or C₁-C₆ branched alkyl; R₃ and R₅ areindependently chosen from a single bond or C₁-C₆ alkyl; R₂ and R₆ areindependently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl, C₃-C₆ cycloalkyl,C₃-C₆ cycloalkenyl, or C₃-C₆ aryl; R₁ and R₇ are independently chosenfrom H, C₁-C₆ alkyl, or C₂-C₆ alkenyl; and R₈, R₉, R₁₀, and R₁₁ are H.

[0113] In some embodiments, the compounds of formula 7, R₁ and R7 areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl, R₄ is C₁-C₆saturated n-alkyl or C₁-C₆ saturated branched alkyl, and R₃ and R₆ areindependently chosen from a single bond or C₁-C₆ saturated n-alkyl.

[0114] In some embodiments, all the nitrogens of the polyamine analogare independently secondary, tertiary, or quarternary amino groups.

[0115] Among polyamine analogs for use in this invention are thosecompounds with a demonstrated ability to modulate naturally occurringpolyamine levels in cells. Without intending to be limited by theory,possible mechanisms include competition in the polyamine synthesispathway; upregulation of polyamine catabolizers such as SSAT; affectingpolyamine metabolism.

[0116] Preferred polyamine analogs include, but are not limited to,1,11-bis(ethyl)norspermine (1,11-bis(ethylamino)-4,8-diazaundecane;BE-3-3-3); 1,8-bis(ethyl)spermindine (BES); 1,12-bis(ethyl)spermine(BESm; DESPM-(N¹, N¹²-diethylsperimine; SunPharm);1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4)(Diethylhomospermine, N¹, N¹⁴-diethylhomospermine; DEHOP or DEHSPM;SunPharm); diethyl-norspermine (DENOP; SunPharm); and1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4).

[0117] In addition, polyamine analogs as described in U.S. Pat. Nos.5,889,061, 5,880,161 and 5,541,230 and in international patentapplication WO 00/66587 may be used in the present invention.

[0118] The polyamine analogs, described above, have been synthesized andscreened in a variety of models of disorders such as cancer. Diamines(e.g., derivatives and/or analogs of putrescine with varying numbers ofmethylene groups in the alkyl chain), triamines (e.g. derivatives and/oranalogs of spermidine with varying numbers of methylene groups in thealkyl chain), tetramines (e.g. derivatives and/or analogs of sperminewith varying numbers of methylene groups in the alkyl chain), andcompounds with five or more amino groups (e.g., not direct derivativesof natural polyamines) can be used in the present invention to modulateneurodegenerative disorders such as ALS.

[0119] The role of putrescine derivatives (i.e., the apoptosis inducerORI-1313) is being explored for their effect on melanoma and leukemia(Porter et al. Cancer Res., 47: 2821-2825(1987)). Through the use ofcompetition experiments with various putrescine analogs, the putrescineand ornithine recognition site on PotE was determined to be located atthe cytoplasmic surface and the vestibule of the pore consisting of 12transmembrane segments. Both uptake and excretion of putrescine arecatalyzed by PotE (Kashiwagi, K. et al., J. Biol. Chem. 275: 36007-36012(2000)).

[0120] A comparison of the structure-activity relationships betweenspermidine and spermine analogues showed that activity is highlydependent on the length of the triamines and the size of theN(alpha),N(omega)-substituents. Various triamines (spermidine analogs)may offer therapeutic advantages over the corresponding tetraamines(spermine analogs) (Bergeron et al. J Med Chem 40(10):1475-1494 (1997)).Studies using fluorinated spermidine analogs illustrate the differentbiological and biochemical properties of these analogs. For example, thedifluorinated spermidine analogues, 7,7-difluorospermidine, was shown tobe capable of repressing ODC and SAM-DC activities, depleting tumorcells of its spermine content, and exerting polyamine antagonist effects(Mamont et al. Adv Exp Med Biol 250:691-706 (1988)).

[0121] In addition, Bums et al. describe the synthesis andcharacterization of spermine/amino acid conjugates which may inhibit theuptake of spermidine into MDA-MB-231 breast cancer cells. One analog,Lys-Spm conjugate, when used in combination with DFMO, was shown toinhibit cytostatic growth of a variety of cancer cells, reduce cellularconcentrations of putrescine and spermidine while not affecting thelevels of spermine (Bums et al. J Med Chem 44(22): 3632-3644 (2001)). Inone embodiment of the present invention, the Lys-Spm conjugate analogmay be used in combination with DFMO to modulate neurodegenerativedisorders, e.g. ALS

[0122] In another aspect, the invention pertains to using spermineanalogs to modify or modulate the polyamine pathway. In a preferredembodiment, the spermine analog is N(1),N(11)-diethylnorspermine(DENSPM). The term “DENSPM” is intended to cover all isotopes andmetabolites of the above compound. DENSPM is intended to cover allpharmaceutically acceptable salts and/or isomeric forms as well asstructurally similar compounds. Non-limiting examples of derivatives canbe found in U.S. Pat. No. 5,962,533, U.S. Pat. No. 5,886,051, U.S. Pat.No. 6,184,232, U.S. Pat. No. 6,342,534, and U.S. Pat. No. 6,235,794.DENSPM has been shown to reduce cellular concentrations of putrescine,spermidine and spermine by down-regulating the activity of the polyaminebiosynthetic enzymes and up-regulating the activity of the catabolicenzyme spermidine/ spermine N(1)-acetyltransferase (SSAT). Studies usingDENSPM in the breast cancer cell line L56Br—C1, have linked DENSPM'sability to increase SSAT activity and inhibit of cell proliferation tothe activation of caspases. (Hegardt et al. “Rapid caspase-dependentcell death in cultured human breast cancer cells induced by thepolyamine analogue N(1),N(11)-diethylnorspermine.” Eur J Biochem269(3):1033-9 (2002)). Phase I clinical trial in patients with advancednon-small cell lung cancer suggest that DENSPM can safely beadministered to patients with minimal toxicity (Hahm et al. “Phase Istudy of N(1),N(11)-diethylnorspermine in patients with non-small celllung cancer.” Clin Cancer Res 8(3):684-90 (2002)).

[0123] Furthermore, Bergeron and co-workers described new synthesismethods for hydroxylated polyamine analogs includingN(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPMENSPM) and the firstsynthesis of (2R,10S)-N(1)-cyclopropylmethyl-2,10-dihydroxy-N(11)-ethylnorspermine[(2R,10S)—(HO)(2)CPMENSPM]. Bergeron et al. concluded that thehydroxylation of norspermine analogues may be improve antiproliferativeactivity while reducing toxicity. These cyclopropyl compounds reducedcellular concentrations of putrescine and spermidine. In addition, thecyclopropyl compounds, as well as DENSPM and (2R,1OR)—(HO)(2)DENSPM,decrease ornithine decarboxylase and S-adenosylmethionine decarboxylaseactivity (Bergeron et al. “Synthesis and evaluation of hydroxylatedpolyamine analogues as antiproliferatives.” J Med Chem 44(15):2451-9(2001)). Therefore, DENSPM derivatives as well as modified DENSPMderivatives (i.e. hydroxylated) may be useful in the present inventionto reduce neurodegenerative disorders.

[0124] In one aspect, the invention pertains to using DFMO to amelioratethe symptoms and onset of ALS. DFMO is an inhibitor of ornithinedecarboxylase, which is the rate limiting enzyme of the polyaminebiosynthetic pathway. DFMO inhibits polyamine synthesis and has beenshown to be effective for cancer prevention in many organ systems,inhibition of cancer growth (U.S. Pat. No. 6,013,646), and reduction oftumor size. DFMO's action is synergistic with other antineoplasticagents. In addition, DFMO has been used as a trypanocidal agent. DFMOhas been used both topically and systemically. Topically DFMO, orVaniqa, has been used to inhibit or slow hair growth. Systemically,DFMO, or Ornidyl, has been used to treat African sleeping sickness, adisease caused by protozoa.

[0125] The terms “DFMO,” and “eflornithine,” as used interchangeablyherein, refer to the compound that is chemically designated as2-(Difuoromethyl)-DL-ornithine, 2-(Difluoromethyl)ornithine,DL-α-difluoromethylornithine, N-Difluoromethylornithine, omidyl, andα,δ-Diamino-α-(difluoromethyl)valeric acid, has a molecular formula ofC₆H₁₂F₂N₂O₂, has a molecular weight of 182.17, and has the followingchemical structure:

[0126] The term “DFMO” is intended to cover all isotopes of the abovecompound.

[0127] Besides the polyamine, spermine or spermidine analogs(collectively referred to as “analogs”) listed above, stereoisomers,salts or protected derivatives thereof, can be used. The invention alsocomprises methods of using an effective amount of any of the analogslisted above, or stereoisomers, salts or protected derivatives thereof(or a composition comprising an effective amount of any of the analogslisted above, or stereoisomers, salts or protected derivatives thereof)in effecting a modulation of the polyamine pathway and ALS. Theinvention also comprises any analog listed above, or stereoisomers,salts or protected derivatives thereof, for use in preparingcompositions (i.e., medicaments) useful for amelioration of ALS.

[0128] Any analog listed above, or stereoisomers, salts or protectedderivatives thereof (or a composition comprising an effective amount ofany polyamine analog listed above, or stereoisomers, salts or protectedderivatives thereof) can be used in vitro or in vivo. For in vitro use,a suitable biological sample (such as a blood sample, which may or maynot be enriched for the abnormal macrophage population) is contactedwith the composition(s). For in vivo use, a composition of the inventionis generally administered according to the manufacturer's/supplier'sinstructions. Generally, the analogs are administered by subcutaneous orintravenous injection. They can also be administered orally.

[0129] The amount of an analog (or stereoisomers, salts or protectedderivatives thereof) to be administered will depend on severalvariables, such as the particular analog (or sterioisomer, salt orprotective derivative) used, the time course of administration, thecondition of the subject, the desired objective, the extent of disease,how many doses will be administered, and whether any other substancesare being administered. In the case of polyamine analogs (orstereoisomer, salt, or protected derivative thereof), the amount willgenerally be between about 1 to about 300 mg/m²/day, possibly betweenabout 15 to about 150 mg/m²/day. Administration is generallyintermittent, meaning that analog (or stereoisomer, salt, or protectedderivative thereof) is administered at a period of at least 1-2 days andthen not administered for a period of at least 1-2 days, with the cyclerepeated as indicated. In one embodiment, the polyamine analog (orstereoisomer, salt, or derivative thereof) is administered for 7 daysfor three weeks, followed by a “a drug holiday” when no analog isadministered.

[0130] Routes of administration will generally depend on the nature ofthe particular polyamine analog (or stereoisomer, salt or protectivederivative) used, and can be, for example, oral or by injection(subcutaneous or intravenous). Administration is generally byintravenous or subcutaneous injection.

[0131] Preferably, an analog (or stereoisomer, salt or protectedderivative), or other modulating agent that interferes with thepolyamine synthetic pathway, polyamine metabolism, and/or theintracellular concentration maintenance of an polyamine, e.g.,putrescine, spermidine, or spermine, is administered in a suitablepharmaceutical excipient. Pharmaceutical excipients are known in the artand are set forth in Remington: The Science and Practice of Pharmacy,20^(th) edition, Mack Publishing (2000). The polyamine analog can alsobe associated with another substance that facilitates agent delivery totarget cell, e.g. macrophages, neurons, microglia, and astrocytes, orincreases specificity of the agent to these target cells. For example,an agent(s) can be associated into liposomes as a means of delivery to atarget cell. Liposomes are known in the art. The liposomes in turn canbe conjugated with targeting substance(s), such as IgGFc receptors.Substances that increase macrophage phagocytosis such as zymosan ortetrachlorodecaoxygen (TCDO) and/or activation such as MCSF, GMCSF orIL-3 may also be used to increase uptake of anti-proliferative agent(s).

[0132] A polyamine, spermidine or spermine analog (or stereoisomer, saltor protected derivative) may be administered alone, or in conjunctionwith other substances and/or therapies, depending on the context ofadministration (i.e., desired end result, condition of the individual,and indications). The phrase “in conjunction with” means that an agentis administered prior to, concurrently, or after other substance ortherapy. Examples of substances that might be administered inconjunction with an agent include, but are not limited to, riluzole(RILUTEK®). Studies with riluzole, approved by the Food and DrugAdministration for therapy of ALS, have demonstrated in statisticallysignificant effects on survival of patients with ALS (Bensimon et al.New Engl. J. Med. 330:585-591 (1994); Lacomblez et al. Lancet347:1425-1431 (1996)). Often ALS therapy includes treatment aimed atcontrol of symptoms. Accordingly, examples of substances for treatmentof symptoms associated with ALS that might be administered inconjunction with an agent include, but are not limited to, baclofen,diazepam, trihexyphenidyl and/or amitriptyline.

[0133] The mechanistic effectiveness of various polyamine, spermidine,or spermine analogs and enzyme inhibitors can be determined in specificcell lines at least in part by their ability to deplete intracellularpolyamine pools. Kramer et al. (Biochem. Pharmacol. 50:1433 (1995))describe the use of 4-fluoro-L-ornithine to monitor metabolic fluxthrough the polyamine biosynthetic pathway. It was determined that themetabolic flux indicated by the rate of appearance of fluorinatedpolyamines, reflected the proliferation status of the cells. U.S. Pat.No. 5,498,522 outlines the use of SSAT enzyme protein, or mRNAtranscripts can be measured directly, or other determinants related toSSAT induction can be measured, such as SSAT co-factor acetylCoA, andthe SSAT products N1-acetylspermine and N1-acetylspermidine. To furtherdetermine the effect of a polyamine analog's administration, anindividual can be monitored for disease (or precursor disease)progression as well as biochemical and/or genetic markers of disease (orprecursor disease). With respect to disease progression, multiple ratingscales (i.e., indices of clinical function) have been established andare known in the art for ALS.

[0134] (ii) Antiproliferative Agents

[0135] Polyamines play a role in cell growth and proliferation and theinduction of cell growth and proliferation has been shown to beassociated with a increase in ODC activity and the correspondingincrease in the level of putrescine as well as the other polyamines. ODCinhibitors have been used in the treatment of cancer in which theypresumably assert their therapeutic effect by blocking polyamineformation, and thereby slowing, interrupting, inhibiting, or stoppingthe cancer cell proliferation and metastases (See US. Pat. Nos.6,277,411 and 4,499,072).

[0136] In recent years growth factors have received a great deal ofattention from the ALS research community as a possible means ofpreventing neuronal death. While neuronal dysfunction or death iscertainly the end result of the ALS disease process, great uncertaintyremains about the causes of neuronal death. It has been largely assumedthat neurons independently become sick or apoptotic. Recent transgenicmouse studies have shown that neuron specific expression of mSOD1 may beinsufficient to cause or accelerate disease. Furthermore, chimericstudies mixing mutant and wild-type SOD mice demonstrate that wild-typeneurons surrounded by mSOD astrocytes and microglia still die. It istherefore likely that neurons must interact with another cell type inorder for disease to occur. While the numbers of viable neurons dropsover the disease course, other cells such as astrocytes and microgliabecome more numerous. Therefore proliferation or activation of thesecell types may be responsible for neuronal damage. Reactiveastrocytosis, microgliosis and activation of microglia are commonhallmarks of many neurodegenerative diseases including ALS. The presentinvention provides methods and screening assays for pharmacologicalagents that are capable of reducing reactive astrocytosis, microgliosis,activation of microglia, macrophage proliferation. Pharmacologicalagents identified through these assays may be useful in treating and/orreducing neurological disorders. Also within the scope is the use ofanti-proliferative agents that can also effect the polyamine pathway,such as hydroxyurea and DFMO to ameliorate ALS. In a preferredembodiment, the anti-proliferative agent is hydroxyurea.

[0137] Hydroxyurea, an agent approved for the treatment of leukemia andovarian cancer and currently being studied as a treatment for HIVdisease, interferes with viral replication by inhibiting the cellularenzyme ribonucleotide reductase. This inhibition results in a reductionof the supply of the deoxyribonucleotides needed to synthesize new DNAresulting in a decrease in cell proliferation. Ribonucleotide reductaseconverts ribonucleotides to deoxyribonucleotides, which is essential inDNA synthesis. Inhibition of ribonucleotide reductase can result fromthe specific interaction of an inhibitor with either of the two subunitsof the enzyme. Hydroxyurea, in addition to thiosemicarbazones,2,3-dihydro-1H-pyrazole[2,3-a]imidazole (IMPY), and several otherantitumor agents, inhibits ribonucleotide reductase through itsinteraction with the smaller subunit (Cory, J. et al. Adv. Enzyme Regul.23, 181-192 (1985)) Hydroxyurea functions at multiple levels to affect anumber of different pathways, for example, hydroxyurea is likely toeffect ODC activity in the polyamine pathway, to inhibit this enzyme.Hydroxyurea is also an anti-proliferative agent that reduces theproliferation of microglia, astrocytes and neurons. The beneficialeffects of DFMO and hydroxyurea are shown in Examples 2, 3 and 6.

[0138] In addition to hydroxyurea, other anti-proliferative agents thatmay affect the polyamine pathway include, but are not limited to,daunomycin, mitomycin C. daunrorubicin, doxorubincin, 5-FU, cytocinearabinoside, colchicine, cytochalasin B, bleomycin, vincristin,vinblastine, methotrexate, cis platinum, ricin, abrin, diptheria toxin,and saporin. These anti-proliferative agents may also function tomodulate macrophages.

[0139] Other suitable agents are those which affect the closelyregulated intracellular concentration of spermidine. An example of suchan agent is MGBG (mitoguazone dihydrochloride; XYRKAMINE®; Ilex, Texas)which inhibits S-adenosylmethionine decarboxylase which in turn isrequired for the production of polyamines. Any agent that interfereswith polyamine interactions with proliferating macrophage target, suchas DNA, RNA, and/or membranes would likewise be suitable. Another typeof useful agent is one that interferes with polyamine interactions withDNA. Such an agent(s) could exert this function, for example, by any ofthe effects above (i.e., interfering with the polyamine syntheticpathway and/or metabolism, disturbing the concentration of intracellularspermine, competitors, etc.) as well as affecting polyamine function interms of interacting with DNA. It is understood that, with respect tothese and any other agent described herein, toxicology considerationsalso must be taken into account when determining whether, and/or in whatamount, an agent is to be used.

[0140] In one embodiment, pharmacological agents that inhibit cellproliferation may be used to modulate, treat, slow, and/or arrestneurodegeneration. In a preferred embodiment, astrocytosis and/ormicrogliosis is inhibited. Administration of more than onepharmacological agent may be beneficial in the treatment of neurologicaldisorders. Administering both a polyamine pathway inhibitor and anantiproliferative agent is within the scope of the present invention.For example, an ODC inhibitor may produce an additive or synergisticeffect with an anti-proliferative agent. Thus, when combinationneurological therapy is used, the dosage of the ODC inhibitor may beless than when used alone. In combination with an ODC inhibitor, theanti-proliferative agent may be administered at a lower dosage or atless frequent intervals compared with use of the anti-proliferativeagent alone.

[0141] (iii) Enzyme Inhibitors

[0142] The enzymes of the polymamine pathway may be reversibly inhibitedthrough the noncovalent binding of an inhibitor of at least one of theenzymes of the pathway. This inhibition may be competitive if theinhibitor binds reversibly to the active site of the enzyme, therebypreventing the native substrate from binding. Additionally, if theinhibitor and substrate bind simultaneously to the enzyme withoutcompeting for the same binding site, the inhibition may benoncompetitive. If the polyamine pathway is inhibited reversibly, theinhibitor may inhibit different enzymes in the pathway leading to adecrease in at least one of the polyamines selected from the groupconsisting of putrescine, spermidine, and spermine. An example of areversible ODC inhibitor is α-methylomithine (T. Thomas et al. Cell.Mol. Life Sci. 58: 244-258 (2001)).

[0143] Enzyme inhibition may also be irreversible. These inhibitors arehighly specific and selective in binding the active site of their targetenzyme. Suicide inhibitors form a class of irreversible inhibitors thatare activated specifically by their target enzyme. DFMO is a suicideinhibitor of ODC. Non-limiting examples of irreversible ODC inhibitorsinclude, but are not limited to, DFMO, α-halomethyl ornithine, methyland ethyl esters of monofluoromethyl dehydroornithine, the R, R-isomerof methyl acetylenic putrescine (i.e., (2R, 5R)-6-heptyne-2,5-diamine),amidinoindan-1-one 2′-amidinohydrazone (CGP 48664), optical isomers andcombinations thereof. In addition, pharmacological agent is alsointended to include other ODC inhibitors with similar structure andfunction to DFMO that are described by the core formulas:

[0144] where X is —CHF₂ or —CH₂F, R is H or COR₁, R₁ is OH or loweralkoxy groups, and the pharmaceutical acceptable salts and isomersthereof. Other inhibitors of ODC known in the art are described in U.S.Pat. Nos. 4,499,072 and 5,002,879; and the work of Bey et al.(“Inhibition of Basic Amino Acid Decarboxylases Involved in PolyamineBiosynthesis,” Inhibition of Metabolism Biological Significance andBasis for New Therapies, McCann et al, eds.; Academic Press, (1987)1-32). Inhibitors of S-adenosyl-L-methionine decarboxylase are describedby Pegg (“Polyamine Metabolism and Its Importance in Neoplastic Growthand as a Target for Chemotherapy,” Cancer Res. 48: 759-774 (1998)) andWilliams-Ashman et al. (“Methylgly-oxal Bis(guanylhydrazone) as a PotentInhibitor of Mammalian and Yesast S-Adenosylmethionine Decarboxylases,”Biochem Biophys. Red. Commun. 46: 288-295 (1972)).

[0145] III. Treatment of Neurodegenerative Disorders Using PolyamineModulating Drugs

[0146] In one aspect, the methods of the invention can be used tomodulate, or ameliorate a neurodegenerative disorder. Theneurodegenerative disorder is selected from the group consisting of ALS,Creutzfelt Jacob's (CJD), Huntington's (HD), Stroke and Alzheimer'sdisease (AD). In a preferred embodiment, the neurodegenerative disorderis ALS. In another aspect, the methods of the invention can be used toscreen for disregulation or abnormal levels of a polyamine such asputraciene, spermidine and spermine. In a preferred embodiment, thepolyamine is putraciene.

[0147] Besides astrocytosis, microgliosis, and inflammation, these samediseases also demonstrate significant regional alterations in polyaminesand polyamine metabolism. For instance, the enzyme ODC is over-expressedin the injured regions of the AD brain. ODC over-expression has beenobserved under basal conditions in the Wobbler mouse, one of the animalmodels of ALS. Human ALS patients show elevated levels of ornithine (thepolyamine precursor) in the spinal tissue and arginase (the enzyme whichconverts arginine to ornithine) in the CSF. Polyamines are integrallyinvolved in cellular proliferation, differentiation signaling, immunecell activation, and cell death. The present invention provides methodsand screening assays associating polyamine deregulation to ALS.Polyamine levels were measured in the brain and spinal cords of SOD1G93A mice demonstrating that polyamines are indeed over expressed.Examples 2 and 3 demonstrate that polyamine synthesis in the G93A mSOD1mouse model of ALS can be inhibited with an ODC inhibitor, DFMO,resulting in delayed onset of disease and extension of the life span ofthe SOD1 G93A mice.

[0148] In one aspect of the invention, the polyamine analogs can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises a polyamine analog and a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of thepharmacological agent.

[0149] The pharmaceutical compositions of this invention can be in avariety of forms. These include, for example, liquid, semi-solid andsolid dosage forms, such as liquid solutions (e.g., injectable andinfusible solutions), dispersions or suspensions, tablets, pills,powders, liposomes and suppositories. The preferred form depends on theintended mode of administration and therapeutic application. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the pharmacological agent is administered by anintraperitoneal injection.

[0150] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., the pharmacological agent) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization.

[0151] Generally, dispersions are prepared by incorporating the activecompound into a sterile vehicle that contains a basic dispersion mediumand the required other ingredients from those enumerated above. In thecase of sterile, lyophilized powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and spray-drying that yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

[0152] The polyamine analog can be administered by a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the polyamine analog can be preparedwith a carrier that will protect the analog against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art. (See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978; U.S. Pat. Nos. 6,333,051 toKabanov et al., and 6,387,406 to Kabanov et al.).

[0153] In certain embodiments, a polyamine analog of the invention canbe orally administered, for example, with an inert diluent or anassimilable edible carrier. The analog (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the analogs may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

[0154] In certain embodiments, a pharmacological agent can beadministered in a liquid form. The pharmacological agent is freelysoluble in a variety of solvents, such as for example, methanol,ethanol, and isopropanol. The pharmacological agent is, however, highlylipophilic and, therefore, substantially insoluble in water. A varietyof methods are known in the art to improve the solubility of thepharmacological agent in water and other aqueous solutions. For example,U.S. Pat. No. 6,008,192 to Al-Razzak et al. teaches a hydrophilic binarysystem comprising a hydrophilic phase and a surfactant, or mixture ofsurfactants, for improving the administration of lipophilic compoundssuch as the pharmacological agent of the present invention.

[0155] Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a pharmacological agent of theinvention is coformulated with and/or coadministered with one or moreadditional therapeutic agents that are useful for improving thepharmacokinetics of the polyamine analog. Methods of improving thepharmacokinetics of the pharmacological agent have been disclosed, forexample, in U.S. Pat. Nos. 6,342,250 to Masters, 6,333,051 to Kabanov etal., 6,395,300 to Straub et al., 6,387,406 to Kabanov et al., and6,299,900 to Reed et al. Masters discloses a drug delivery device andmethod for the controlled release of pharmacologically active agents andthe same methodology maybe used for the polyamine analogs of the presentinvention. The drug delivery device disclosed by Masters is a filmcomprising one or more biodegradable polymeric materials, one or morebiocompatible solvents, and one or more pharmacologically active agentsdispersed uniformed throughout the film. In U.S. Pat. No.6,333,051,Kabanov et al. disclose a copolymer networking having at least onecross-linked polyamine polymer fragment, at least one nonionicwater-soluble polymer fragment, and at least one suitable biologicalagent, including the pharmacological agent of the present invention.According to the teachings of this patent, this network, referred to asa nanogel network, improves the therapeutic effect of thepharmacological agent by decreasing side effects and increasingtherapeutic action. In another patent, U.S. Pat. No. 6,387,406, Kabanovet al. also disclose another composition for improving the oral deliveryof numerous pharmacological agents. This delivery vehicle comprises abiological agent and a poly(oxyehtylene)-poly(oxypropylene) blockcopolymer. Straub et al. disclose porous drug matrices for use withdrugs, and in particular, for use with low-aqueous solubility drugs, forenhancing solubility of the drug in an aqueous solution. Reed et al.disclose a drug delivery system, which uses a dermal penetrationenhancer to transport a variety of physiologically active agents,including the pharmacological agent of the present invention, across adermal surface or mucosal membrane of a subject.

[0156] Other methods for improving the delivery and administration ofthe pharmacological agent e.g., a polyamine analog include means forimproving the ability of the pharmacological agent to cross membranes,and in particular, to cross the blood-brain barrier. In one embodiment,the pharmacological agent can be modified, e.g., made hydrophobic, toimprove its ability to cross the blood-brain barrier, and in analternative embodiment, the pharmacological agent can be co-administeredwith an additional agent, such as for example, an anti-fungal compound,that improves the ability of the pharmacological agent to cross theblood-brain barrier. Alternatively, precise delivery of thepharmacological agent into specific sites of the brain, can be conductedusing stereotactic microinjection techniques. For example, the subjectbeing treated can be placed within a stereotactic frame base(MRI-compatible) and then imaged using high resolution MRI to determinethe three-dimensional positioning of the particular region to betreated. The MRI images can then be transferred to a computer having theappropriate stereotactic software, and a number of images are used todetermine a target site and trajectory for pharmacological agentmicroinjection. The software translates the trajectory intothree-dimensional coordinates that are precisely registered for thestereotactic frame. In the case of intracranial delivery, the skull willbe exposed, burr holes will be drilled above the entry site, and thestereotactic apparatus used to position the needle and ensureimplantation at a predetermined depth. The pharmacological agent can bedelivered to regions, such as the cells of the spinal cord, brainstem,or brain that are associated with the disease or disorder. For example,target regions can include the medulla, pons, and midbrain, cerebellum,diencephalon (e.g., thalamus, hypothalamus), telencephalon (e.g., corpusstratium, cerebral cortex, or within the cortex, the occipital,temporal, parietal or frontal lobes), or combinations, thereof.

[0157] In one aspect of the invention, the pharmacological agent doesnot cross the blood brain barrier. The proliferating macrophages migrateto the brain leading to the production of microglia and/or astrocytes.Thus, inhibition or suppression of proliferative of macrophagesperipherally may prevent or slow neurological disease progressionthereby eliminating the need for the pharmacological agent to act in thebrain.

[0158] In yet another aspect of the invention, the blood brain barriermay be compromised by the elevated production of polyamines associatedwith neurological disorder such that an effective amount of thepharmacological agent may be able to reach its target site. Polyamineshave been shown to play a role in the disruption of the blood-brainbarrier (BBB) in various pathological states suggesting that polyaminesmay play a role as mediators of vasogenic edema formation in the brainfollowing brain injuries (Glantz L et al. J Basic Clin Physiol Pharmacol7:1-10 (1996)).

[0159] Pharmacological agents can be used alone or in combination totreat neurodegenerative disorders. For example, the pharmacologicalagent can be used in conjunction with polyamine analogs or other ODCinhibitors, for example, to produce a synergistic effect. Likewise, thepharmacological agent can be used alone or in combination with anadditional agent, e.g., an agent which imparts a beneficial attribute tothe therapeutic composition, e.g., an agent which effects the viscosityof the composition. The combination can also include more than oneadditional agent, e.g., two or three additional agents if thecombination is such that the formed composition can perform its intendedfunction.

[0160] The pharmaceutical compositions of the invention can include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a pharmacological agent of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the pharmacological agent can varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the pharmacological agent to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thepharmacological agent are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0161] Dosage regimens can be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response). For example, asingle bolus can be administered, several divided doses can beadministered over time or the dose can be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0162] An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a pharmacological agent of theinvention is between 100 mg/Kg/day and 10,000 mg/Kg/day, administered toa subject. Preferably, administration of a therapeutically effectiveamount of pharmacological agent results in a concentration ofpharmacological agent in the bloodstream that is between about 0.1 μMand 1000 μM. Preferably, the concentration of pharmacological agent inthe blood is between about 1-100 μM. More preferably, the concentrationof pharmacological agent in the blood is between about 1-50 μM. It is tobe noted that dosage values can vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

[0163] IV. Screening Assay for Pharmacological Agents

[0164] The methods of the invention can be used to screen a large numberof candidate compositions to find pharmacological agents capable ofmodulating polyamine levels in a subject. The screening assay methods ofthe present invention are preferably cellular assays that include a cellline that can be stably cultured using standard cell culture techniquesknown to those having ordinary skill in the art. For example, thescreening assay methods of the present invention can include the stepsof (i) determining the polyamine level in a substrate, (ii) applying apharmacological agent to the substrate, and (iii) measuring changes inthe polyamine level in response to the applied pharmacological agent.

[0165] In one embodiment, the assay will identify pharmacological agentsthat decrease polyamine levels in proliferating immune cells of alltypes. In another embodiment, assays and methods are disclosed forscreening for pharmacological agents that are capable of reducing thelevels of polyamine levels in the brain and spinal cord of neurologicalafflicted subjects. The method comprises obtaining non-transgenicwild-type mice as well as symptomatic and asymptomatic G93A SOD1 mice,administering a pharmacological agent and a control to a group of eachmouse population, sacrificing the mice, homogenizing the brain andspinal cord, quantitating the level of polyamine levels present in eachtissue, and comparing the polyamine levels between the mousepopulations. Pharmacological agents that are found to decrease polyaminelevels when compared to the control population are possible drugcandidates.

[0166] The term “measuring the difference in the polyamine level” or“measuring the difference in polyamine expression,” as used herein,refers to any means or methods of comparison between the level ofpolyamine activity in a substrate prior to the application of apharmacological agent and the level of polyamine application afterapplication of the pharmacological agent. A statistically significantdifference in the polyamine level can be a difference in values by afactor of 10 between the test sample and the control sample, morepreferably, a difference by a factor of 8, even more preferably, adifference by a factor of 6, even more preferably, a difference by afactor of 4, and most preferably, a difference by a factor of 2.

[0167] The screening assay method of the present invention can be rununder normal conditions, or alternatively, the screening assay can berun in the presence various stress models. Preferable stress modelsinclude, but are not limited to, heat shock models and oxidative stressmodels. According to the present invention, the stress models can beintroduced either before or after the application of a pharmacologicalagent to the substrate.

Equivalents

[0168] Those skilled in the art will appreciate, or be able to ascertainusing no more than routine experimentation, further features andadvantages of the invention based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references are herein expresslyincorporated by reference in their entirety.

EXAMPLES

[0169] The following examples illustrate that the pharmacological agentof the present invention, e.g., DFMO and DENSPM, delay the diseaseprogression and prolongs the life of male SOD1 G93A mice. The beneficialeffect of DFMO in the SOD1 G93A mouse model demonstrates that thepolyamine biosynthetic pathway is a novel target for drug discovery inALS. The following examples are merely illustrative of the presentinvention and should not be construed so as to limit the scope of thisinvention.

Example 1

[0170] Methods and Materials

[0171] (i) Model Description: The SOD1 G93A (high copy) mouse model wasused as the model for ALS. This mouse model carries 25 copies of thehuman G93A SOD mutation and is driven by the endogenous promoter.Survival in the mouse is copy dependent. The high copy G93A has a mediansurvival of around 128 days. High molecular weight complexes of mutantSOD protein are seen in the spinal cord beginning around day 30. At day60 reactive astrocytosis (GFAP reactive) are observed; activatedmicroglia are observed from day 90 onwards. Studies by Gurney et al.showed that at day 90 reactive astrocytosis loses statisticalsignificance while microglial activation is significantly elevated andcontinues to be elevated through the end stage of the disease.

[0172] Many drugs that have shown efficacy in this model have moveforward into human clinical trials based on the data resulting formstudies with this model. Experience with riluzole, the only approveddrug in the treatment of ALS, indicates that the mouse ALS model is agood predictor of clinical efficacy.

[0173] (ii) Murine FALS model: Heterozygous transgenic mice carrying thehuman SOD-1 (G93A) gene were obtained from Jackson Laboratory (BarHarbor, Me., USA).

[0174] (iii) Pharmacological agent: Difloromethylornithine (DFMO) is anirreversible inhibitor of the enzyme ornithine decarboxylase (ODC), therate-limiting enzyme in polyamine biosynthesis. DFMO is approved asOrnidyl for the treatment of African Sleeping Sickness. DFMO wasobtained in oral form from ILEX Oncology, Inc (San Antonio, Tex.).

[0175] (iv) Methods: Forty (n=40) SOD1 G93A high copy mice obtained fromJackson Laboratory (Bar Harbor, Me.) were separated into two groups oftwenty (n=20) with an equal number of males (n=10) and females (n=10) ineach group. The groups were also litter matched to reduce groupvariation. Male animals were singly housed and females were group housedto conserve space. Animals received standard rodent chow and water whilethey were able to feed themselves. When animals had difficulty in movingand taking care of themselves chow and water were replaced with foodpellets in bedding and Jell-O for water.

[0176] (v) Pharmacological Agent Delivery: All pharmacological agentswere administered to SOD1 G93A mice. DFMO was delivered according to achronic delivery protocol applied in the human glioblastoma trial (UCSFClinical trial protocol of ALS).

[0177] In Example 2, the animals were dosed at 3200 mg/Kg/day of DFMO indrinking water for 21 days followed by a 7 day drug holiday (no DFMO).In Example 3, the animals were continually dosed at 3200 mg/Kg/day ofDFMO in drinking water. Controls animals were given water as vehicle.All treatments are performed 7 days per week and treatment was initiatedat day 60 of life. The treated group continued to receive 3200 mg/kg/dayof DFMO in drinking water until a neurological score of 2 (See section(vi) below) at which point drug was delivered via intraperitinealinjection (IP) injection. When a neurological score of 2 is attained,the animals are no longer capable of drinking themselves due toparalysis. At this point, the same dosage of DFMO is administered to theanimal in IP injection form. Controls were given water as vehicle untila neurological score of 2 at which point they were injected with salineas vehicle.

[0178] In Example 6, hydroxyurea was administered by intraperitinealinjection at a dose of 250 mg/Kg/day once daily for 7 days a week,beginning at day 50 until day 110. The treatment was discontinued afterday 110 due to toxicity arising from the hydroxyurea. Despite, thetoxicity related to the drug, the hydroxyurea still continued to providea protective effect in terms of prolonging the lifespan of the animal.Cytotoxicity resulting from the drug can readily be identified from theprotective effects of the drug by observing signs of toxicity such asneutrophenia, thrombocytopenia and haemolysis. The cytotoxic effects ofhydroxyurea can be reduced by altering and optimizing the dose of thedrug that is administered to the animal.

[0179] In Example 7, DENSPM was administered by intraperitinealinjection at a dose of 24 mg/kg/day twice daily for 7 days a week tilldeath.

[0180] (vi) Neurological Scoring: Neurological score of each limb wasmonitored and recorded according to a defined 4-point scale definedbelow:

[0181] 0=Normal reflex on the hind limbs (animal will splay its hindlimbs when lifted by its tail).

[0182] 1=Abnormal reflex of hind limbs (lack of splaying of hind limbswhen animal is lifted by the tail).

[0183] 2=Abnormal reflex of limbs and evidence of paralysis.

[0184] 3=Lack of reflex and complete paralysis.

[0185] 4=Inability to right themselves when placed on the sides in 30seconds or found dead. The animals are sacrificed at this stage ifalive.

[0186] (vii) End Points and Statistics: The primary end point issurvival with secondary end points of neurological score and bodyweight. Neurological score observations and body weight are made andrecorded five days per week.

[0187] (viii) Statistical Analysis: Statistical analysis on body weightand neurological score was performed using mixed linear model repeatedmeasure ANOVA. Survival analysis was performed by Kaplan-Meier analysisusing the frailty model for sex and litter correction.

Example 2

[0188] Treatment of ALS in a SOD-1 (G93A) Mouse Model with DiscontinuousDosing of DFMO

[0189] During this study, DFMO was delivered chronically at a dose of3200 mg/Kg/day in drinking water for 21 days followed by a 7 day drugholiday (no drug) according to the methods described in Example 1.Results are shown in FIGS. 2 and 3 and described below.

[0190] Percent Survival Analysis: In the females 70% of the deaths ofanimals in the treatment group occurred during the time of drug holidayor a few days immediately succeeding the holiday. However, only 30% ofdeaths occurred in the control group during the same period (FIG. 2).DFMO, a substrate analog inhibitor of ornithine decarboxylase delays theonset of disease and extends the survival of the SOD1 G93A mouse.

[0191] Neurological Score analysis: No effect was seen in the combinedresults or in the male mice (data not shown). However, discontinuousdelivery of DFMO was protective in the female SOD1 G93A mouse during thecourse of drug delivery period. The total female effect was 3.5% asshown in FIG. 3. The female mice showed a positive trend sufficient towarrant screening DFMO again with no drug holiday (shown in Example 3).The lack of effect observed in this study in the male mice may be areflection of the varying amount of polyamines between differentgenders. For example, a higher base level of polyamines in males mayrequire a higher or longer dosage of DFMO before the positive effect ofthe pharmacological agent can be observed. This hypothesis is beingexplored in ongoing studies.

Example 3

[0192] Treatment of ALS in a SOD-1 (G93A) Mouse Model with ContinuousDosing of DFMO

[0193] During this study, DFMO was delivered chronically at a dose of3200 mg/Kg/day in drinking water for 21 days without any drug holidayuntil death according to the methods described in Example 1. Results areshown in FIGS. 4-9 and described below.

[0194] Percent Survival Analysis: DFMO extended the life span of theSOD1 G93A mice as shown in FIGS. 4, 6 and 8. DFMO, a substrate analoginhibitor of ornithine decarboxylase delays the onset of disease andextends the survival of the SOD1 G93A mouse.

[0195] Neurological Score analysis: Continuous delivery of DFMO wasprotective in the combined results of the male and female SOD1 G93A(FIGS. 5) as well as in the male (FIG. 7) and female (FIG. 9) SOD1 G93Apopulation. The total combined effect was 9.69% (p=0.02) when analyzedby Cox proportional hazard using litter effect as a frailty term. Themale mice showed an 8.21% effect while the female mice showed an 11.48%effect. Thus, when DFMO was delivered continuously, a positive trend wasobserved regardless of gender.

[0196] Collectively, these results demonstrate that the polyamineputrescine is high in female mice and that inhibiting putrescinesynthesis with DFMO has a beneficial and reproducible effect. Studiesare currently underway to analyze polyamines in male vs. female mSOD1mice and wild-type mice. While positive effects are seen in male mice,the effect is less reliable at current doses. Differences in polyaminesynthesis and regulation exist between the sexes and may account for thedifferent observed effects. The positive male trend in the ongoing fPinjection study using 3200 mg/kg of DFMO, suggests that this may betrue. Higher plasma concentration of DFMO can be achieved by systemicdelivery as opposed to oral delivery.

[0197] In conclusion, ODC inhibitor Eflomithine (DFMO) treated animalsshow signs of improved survival. These data demonstrate a target in ALSthat can be exploited for drug development.

Example 4

[0198] In Vitro Analysis of Modulation of Cell Proliferation inNeurodegenerative Disorders

[0199] This example demonstrates how cells can be monitored and screenedfor a decrease in proliferation following the addition of apharmacological agent. One non-limiting example of a proliferation assaythat may be performed is described. Cells can be seeded in culturedishes, induced to proliferation, treated with either a pharmacologicalagent or PBS, and allowed to progress in the cell cycle. Following adetermined amount of time, [³H]thymidine is added to the medium. After 1hour at 37° C., the cells are washed with PBS and the radioactivethymidine incorporation in cellular DNA was quantified by liquidscintillation counting. Alternatively cell proliferation can be measuredby detecting proliferation marker(s) and/or the uptake of substancessuch as [³H]thymidine, BrdU, and tetrazolium salts (e.g., MTT and XTT).

Example 5

[0200] In Vitro Analysis of Modulation ofPolyamine levels inNeurodegenerative Disorders

[0201] This example demonstrates how cells can be monitored for adecreased levels of polyamines following the addition of apharmnacological agent. The following are non-limiting examples ofassays for measuring polyamine levels that may be performed.

[0202] Cells can be treated as described above for the proliferationassay (See Example 4). The cells can then be harvested and the cellpellets are acidified and sonicated. The solution can be incubated onice and centrifuged to remove the precipitated proteins. Intracellularpolyamine levels can be determined by known HPLC techniques as describedpreviously by (Thomas et al. Breast Cancer Research and Treatment 39:293-306 (1996)).

[0203] Alternatively, brain and spinal cord tissue can be collected fromsymptomatic SOD1 G93A, pre-symptomatic SOD1 G93A, and non-transgenic(wild-type) mice. The tissue can be homogenized in acid containing aninternal standard (i.e. diamioheptane or 1,6-diaminohexane),centrifuged, and stored frozen prior to analysis. Polyamines can then bequantitated by known HPLC techniques of the DANSYL derivatives withfluorescence detection as described above.

Example 6

[0204] Treatment of ALS in a SOD-1 (G93A) Mouse Model with Hydroxyurea

[0205] This example demonstrates the in vivo protective effect ofhydroxyurea in ALS using SOD-1 (G93A) mice. During this study,hydroxyurea was given by intraperitineal injection at 250 mg/Kg/day oncedaily for 7 day a week from day 50 until day 110 days as described inExample 1. The treatment was discontinued after day 110 due to toxicityarising from the hydroxyurea. Despite, the toxicity related to thehydroyurea, the hydroxyurea still continued to provide a protectiveeffect in terms of prolonging the lifespan of the animal. Cytotoxicityresulting form the drug can readily be identified from the protectiveeffects of the drug by observing signs of toxicity such as neutrophenia,thrombocytopenia and haemolysis. The cytotoxic effects of hydroxyureacan be reduced by altering and optimizing the dose of the drug that isadministered to the animal. The results are shown in FIGS. 10 and 11 anddescribed below.

[0206] Percent Survival Analysis: Hydroxyurea extended the life span ofthe SOD1 G93A mice as shown in FIG. 10. The effects were statisticallyand quantitativley similar to those observed with animals treated withDFMO (See Example 2 and 3). Hydroxyurea delays the onset of ALS diseaseand extends the survival of the SOD1 G93A mouse. Hydroxyurea is ananti-proliferative agent, a ribonucleotide reductase inhibitor, and mayalso be involved in the polyamine pathway, possibly acting to inhibitODC.

[0207] Neurological Score analysis: Delivery and treatment withhydroxyurea was protective in the female (FIG. 11) SOD1 G93A population.These results demonstrate that the hydroxyurea may be inhibiting neuronsform proliferating, in particular by reducing proliferation ofastrocytes and microglia. Similar results are anticipated in male SOD1G93A mice.

Example 7

[0208] Treatment of ALS in a SOD-1 (G93A) Mouse Model with DENSPM

[0209] This example demonstrates the in vivo protective effect of DENSPMin ALS using SOD-1 (G93A) mice. During this study, DENSPM was deliveredthrough intraperitoneal injection at 24 mg/kg/day twice daily for 7 daysa week, till death according to the methods described in Example 1.Results are shown in FIGS. 12 and 13 and described below.

[0210] Percent Survival Analysis: DENSPM, a polyamine analog withanti-proliferative activity, delays the onset of disease and extends thesurvival of female SOD1 G93A mouse, as shown in FIG. 12. DENSPM mimicsthe polyamine, operating in a negative feedback mechanism to trick thecell into “thinking” there is sufficient levels of the polyaminepresent, thus shutting down the polyamine synthesis pathway. Similarresults are expected in male SOD1 G93A mice.

[0211] Neurological Score analysis: Delivery of DENSPM was protective inthe female SOD1 G93A population (FIGS. 13). Similar results are expectedin male SOD1 G93A mice.

[0212] Collectively, these results demonstrate that the symptoms of ALScan be modulated by administering modulating agents. In particular, theresults show that the pharmacological agent, DFMO, inhibits the ODCenzyme and affects the levels of putrescine; the polyamine analog,DENSPM, which regulates a negative feedback mechanism tricking the cellto stop synthesizing polyamines; and hydroxyurea, which acts via anunknown mechanism to inhibit ODC, ribonucleotide reductase, and cellproliferation. All of the modulating agents appear to affect thepolyamine biosynthesis pathway and provide protection against ALS.

What is claimed is:
 1. A method for modulating polyamine pathwayactivity in a subject comprising administrating a therapeuticallyeffective amount of a difuoromethylornithine (DFMO) to the subject. 2.The method of claim 1, wherein the DFMO is a racemic mixture of (D)- and(L)-DFMO.
 3. The method of claim 1, wherein the DFMO is (D)-DFMO.
 4. Themethod of claim 1, wherein the DFMO is (L)-DFMO.
 5. A method ofinhibiting progression of amyotrophic lateral sclerosis (ALS) comprisingadministering to a subject thereof a pharmaceutical compositioncomprising a therapeutically effective amount of difuoromethylornithine(DFMO), or pharmaceutically acceptable salts thereof, to therebymodulate the polyamine pathway and ameliorate the progression of ALS. 6.The method of claim 5, wherein the DFMO is a racemic mixture of (D)- and(L)-DFMO.
 7. The method of claim 5, wherein the DFMO is (D)-DFMO.
 8. Themethod of claim 5, wherein the DFMO is (L)-DFMO.
 9. The method of claim5, wherein DFMO is administered via an oral route.
 10. The method ofclaim 5, wherein DFMO is administered via an intravenous route.
 11. Themethod of claim 5, wherein the administered amount of DFMO is between100 mg/Kg/day and 10,000 mg/Kg/day.
 12. The method of claim 5, whereinthe administered amount of DFMO is adjusted based on patient response orplasma levels of DFMO.